Human reticulocalbin isoforms

ABSTRACT

The invention provides two human reticulocalbin isoforms designated individually as RCN γ and RCN δ and collectively as RCN, and polynucleotides which identify and encode RCN. The invention also provides expression vectors, host cells, agonists, antibodies and antagonists. The invention also provides methods for treating disorders associated with expression of RCN.

[0001] This application is a divisional application of U.S. applicationSer. No. 09/270,270, filed Mar. 16, 1999, which is a divisionalapplication of U.S. application Ser. No. 08/910,927, filed Aug. 8, 1997,now U.S. Pat. No. 5,976,801, the contents all of which are herebyincorporated by reference.

FIELD OF THE INVENTION

[0002] This invention relates to nucleic acid and amino acid sequencesof human reticulocalbins and to the use of these sequences in thediagnosis, prevention, and treatment of infectious, developmental,neoplastic, and immunological disorders.

BACKGROUND OF THE INVENTION

[0003] The endoplasmic reticulum (ER) is a system of intracellularmembranes in which protein synthesis and other important metabolicprocesses take place. In cells where the major function of theendoplasmic reticulum is protein synthesis, this organelle contains alarge number of ribosomes and is known as the rough endoplasmicreticulum. Endoplasmic reticulum that is engaged primarily in steroidhormone biosynthesis and contains few ribosomes is known as the smoothendoplasmic reticulum.

[0004] The ER also serves as an intracellular store of Ca²⁺([Ca²+]_(i)).Following stimulation by second messenger molecules, such asinositoltrisphosphate, [Ca²+]_(i) is briefly released from the ER intothe surrounding cytoplasm. Increased levels of [Ca²+]_(i) in thecytoplasm activate a number of enzymatic processes, some of whichcontribute to cell cycle events, and/or to cellular differentiation.Similar processes take place in the dividing cell nucleus duringbreakdown of the nuclear membrane and segregation of chromatids atanaphase.

[0005] In muscle, the ER is termed the sarcoplasmic reticulum (SR) andis the principal source of [Ca²+]₁, which drives muscle contraction.[Ca²+]₁ binds to calmodulin (CaM), which activates CaM protein kinase.CaM protein kinase then phosphorylates light-chain myosin. In relaxedmuscle, myosin is prevented from interacting with actin by tropomyosin.Ca²⁺ binds tropomyosin, causing a conformational change that leads tothe release of actin. Phosphorylated myosin interacts with actin,forming actinomyosin, and the contraction process is initiated. Musclerelaxation is brought about by active transport of Ca²⁺ into the SR by acalcium ATPase pump.

[0006] The calcium-binding domain of many proteins contains the highaffinity Ca²⁺-binding motif often referred to as the EF-hand(Kretsinger, R. H. and Nockolds, C. E. (1973) J. Biol. Chem.248:3313-3326). The EF-hand is characterized by a twelve amino acidresidue-containing loop, flanked by two a-helices, orientatedapproximately 90° with respect to one another. Aspartate (D) andglutamate (E) residues are usually found at positions 10 and 21,respectively, bordering the twelve amino acid loop. In addition, aconserved glycine residue in the central portion of the loop is found inmost Ca²⁺-binding EF-hand domains. Oxygen ligands within this domaincoordinate the Ca²⁺ ion.

[0007] Numerous soluble proteins are retained within the ER by aspecific retrieval receptor which recognizes a C-terminal tetrapeptide,defined as Lys/His-Asp-Glu-Leu (K/HDEL). ER soluble proteins includeendoplasmin, BiP, PDI, and calrecticulin. Calrecticulin is believed tobe the major Ca²⁺-storage protein of the ER; the other three proteinsare involved in folding and maturation of secretory proteins. All fourproteins bind Ca²⁺, but none are members of the EF-hand family (Weis, K.et al. (1994) J. Biol. Chem. 269:19142-19150).

[0008] Novel endoplasmic reticulum Ca²⁺-binding proteins have also beenidentified. Human reticulocalbin is an ER luminal protein isolated froma transitional carcinoma cell line. The protein has six repeats of adomain containing the EF-hand domain, an HDEL C-terminal tetrapeptide,and binds Ca²⁺. A conserved glycine residue in the central portion ofthree of the EF-hand domains is absent, suggesting that humanreticulocalbin plays some role(s) besides Ca²⁺-binding (Ozawa, M. (1995)J. Biochem. (Tokyo) 117:1113-1119). A similar protein, ERC-55, has beenisolated from HeLa cells. It has six EF-hand repeats and an HDELC-terminal peptide, and Ca²⁺. The conserved glycine residue is absentfrom three of the ERC-55 EF-hand domains (Weis, K. et al. (supra)). Bothproteins are expressed ubiquitously, particularly in heart, placenta,lung, and skeletal muscle (human reticulocalbin) and in kidney andskeletal muscle (ERC-55) (Ozawa, M. and Muramatsu, T. (supra); Weis, K.et al. (supra)).

[0009] The human gene encoding human reticulocalbin has been localizedto a region on chromosome 11 (11p13). The gene is hemizygously deletedin individuals with the Wilms tumor, aniridia, genitourinary anomalies,mental retardation (WAGR) syndrome. The homologous murine reticulocalbingene maps to a region of conserved synteny on mouse chromosome 2 and isdeleted in the Small eye Harwell (Sey^(H)) mutation. Loss of the murinereticulocalbin gene could contribute to the early lethality of Sey^(H)and Sey^(Dey) homozygotes (Kent, J. et al. (1997) Genomics 42:260-267).

[0010] Overexpression of reticulocalbin mRNA has been associated withthe increased matrigel invasive properties of three human breast cancercell lines. Conversely, reticulocalbin was not found to be expressed intwo poorly invasive breast cancer cell lines (Liu, Z. et al. (1997)Biochem. Biophys. Res. Comm. 231:283-289).

[0011] An endogenous monoclonal antibody (MAb), which promotes centralnervous system remyelination in a mouse model of multiple sclerosis,specifically reacts with nine independent neonatal rat brain cDNAclones. Five of the clones are identical or highly similar to knowncDNAs or proteins. One of the unknown clones (REM#1) encodes a 98 aminoacid truncated protein (Asakura, K. et al. (1996) J. Neuroimmunol.65:11-19). The MAb immunostains brain, spinal cord, heart, liver,kidney, stomach, erythrocytes, and small intestine. In particular,immunoreactivity is observed for both surface and cytoplasmicdeterminants in glial cells and in dendritic cells of the spleen,thymus, and lymph node (Miller, D. J. et al. (1994) J. Neurosci.14:6230-6238).

[0012] The discovery of new human reticulocalbin isoforms and thepolynucleotides encoding them satisfies a need in the art by providingnew compositions which are useful in the diagnosis, prevention andtreatment of infectious, developmental, neoplastic, and immunologicaldisorders.

SUMMARY OF THE INVENTION

[0013] The invention features substantially purified polypeptides, humanreticulocalbin isoforms (designated collectively as RCN and individuallyas RCN γ and RCN δ) having the amino acid sequence shown in SEQ ID NO:1or SEQ ID NO:3, respectively, or fragments thereof.

[0014] The invention further provides an isolated and substantiallypurified polynucleotide sequence encoding the polypeptide comprising theamino acid sequence of SEQ ID NO:1 or fragments thereof and acomposition comprising said polynucleotide sequence. The invention alsoprovides a polynucleotide sequence which hybridizes under stringentconditions to the polynucleotide sequence encoding the amino acidsequence SEQ ID NO:1, or fragments of said polynucleotide sequence. Theinvention further provides a polynucleotide sequence comprising thecomplement of the polynucleotide sequence encoding the amino acidsequence of SEQ ID NO:1, or fragments or variants of said polynucleotidesequence.

[0015] The invention also provides an isolated and purified sequencecomprising SEQ ID NO:2 or variants thereof. In addition, the inventionprovides a polynucleotide sequence which hybridizes under stringentconditions to the polynucleotide sequence of SEQ ID NO:2. The inventionalso provides a polynucleotide sequence comprising the complement of SEQID NO:2, or fragments or variants thereof.

[0016] The present invention further provides an expression vectorcontaining at least a fragment of any of the claimed polynucleotidesequences. In yet another aspect, the expression vector containing thepolynucleotide sequence is contained within a host cell.

[0017] The invention also provides a method for producing a polypeptidecomprising the amino acid sequence of SEQ ID NO:1 or a fragment thereof,the method comprising the steps of: a) culturing the host cellcontaining an expression vector containing at least a fragment of thepolynucleotide sequence encoding RCN γ under conditions suitable for theexpression of the polypeptide; and b) recovering the polypeptide fromthe host cell culture.

[0018] The invention also provides a pharmaceutical compositioncomprising a substantially purified RCN γ having the amino acid sequenceof SEQ ID NO:1 in conjunction with a suitable pharmaceutical carrier.

[0019] The invention also provides a purified antagonist of thepolypeptide of SEQ ID NO:1. In one aspect the invention provides apurified antibody which binds to a polypeptide comprising the amino acidsequence of SEQ ID NO:1.

[0020] Still further, the invention provides a purified agonist of thepolypeptide of SEQ ID NO:1.

[0021] The invention also provides a method for treating or preventingan infectious disorder comprising administering to a subject in need ofsuch treatment an effective amount of a pharmaceutical compositioncomprising purified RCN γ.

[0022] The invention also provides a method for treating or preventing adevelopmental disorder comprising administering to a subject in need ofsuch treatment an effective amount of a pharmaceutical compositioncomprising purified RCN γ.

[0023] The invention also provides a method for treating or preventing aneoplastic disorder comprising administering to a subject in need ofsuch treatment an effective amount of a purified antagonist to RCN γ.

[0024] The invention also provides a method for treating or preventingan immunological disorder comprising administering to a subject in needof such treatment an effective amount of a purified antagonist to RCN γ.

[0025] The invention also provides a method for detecting apolynucleotide which encodes RCN γ in a biological sample comprising thesteps of: a) hybridizing the complement of the polynucleotide sequencewhich encodes SEQ ID NO:1 to nucleic acid material of a biologicalsample, thereby forming a hybridization complex; and b) detecting thehybridization complex, wherein the presence of the complex correlateswith the presence of a polynucleotide encoding RCN γ in the biologicalsample. In one aspect the nucleic acid material of the biological sampleis amplified by the polymerase chain reaction prior to hybridization.

[0026] The invention further provides an isolated and substantiallypurified polynucleotide sequence encoding the polypeptide comprising theamino acid sequence of SEQ ID NO:3 or fragments thereof and acomposition comprising said polynucleotide sequence. The invention alsoprovides a polynucleotide sequence which hybridizes under stringentconditions to the polynucleotide sequence encoding the amino acidsequence SEQ ID NO:3, or fragments of said polynucleotide sequence. Theinvention further provides a polynucleotide sequence comprising thecomplement of the polynucleotide sequence encoding the amino acidsequence of SEQ ID NO:3, or fragments or variants of said polynucleotidesequence.

[0027] The invention also provides an isolated and purified sequencecomprising SEQ ID NO:4 or variants thereof. In addition, the inventionprovides a polynucleotide sequence which hybridizes under stringentconditions to the polynucleotide sequence of SEQ ID NO:4. The inventionalso provides a polynucleotide sequence comprising the complement of SEQID NO:4, or fragments or variants thereof.

[0028] The present invention further provides an expression vectorcontaining at least a fragment of any of the claimed polynucleotidesequences. In yet another aspect, the expression vector containing thepolynucleotide sequence is contained within a host cell.

[0029] The invention also provides a method for producing a polypeptidecomprising the amino acid sequence of SEQ ID NO:3 or a fragment thereof,the method comprising the steps of: a) culturing the host cellcontaining an expression vector containing at least a fragment of thepolynucleotide sequence encoding RCN δ under conditions suitable for theexpression of the polypeptide; and b) recovering the polypeptide fromthe host cell culture.

[0030] The invention also provides a pharmaceutical compositioncomprising a substantially purified RCN δ having the amino acid sequenceof SEQ ID NO:3 in conjunction with a suitable pharmaceutical carrier.

[0031] The invention also provides a purified antagonist of thepolypeptide of SEQ ID NO:3. In one aspect the invention provides apurified antibody which binds to a polypeptide comprising the amino acidsequence of SEQ ID NO:3.

[0032] Still further, the invention provides a purified agonist of thepolypeptide of SEQ ID NO:3.

[0033] The invention also provides a method for treating or preventingan infectious disorder comprising administering to a subject in need ofsuch treatment an effective amount of a pharmaceutical compositioncomprising purified RCN δ.

[0034] The invention also provides a method for treating or preventing adevelopmental disorder comprising administering to a subject in need ofsuch treatment an effective amount of a pharmaceutical compositioncomprising purified RCN δ.

[0035] The invention also provides a method for treating or preventing aneoplastic disorder comprising administering to a subject in need ofsuch treatment an effective amount of a purified antagonist to RCN δ.

[0036] The invention also provides a method for treating or preventingan immunological disorder comprising administering to a subject in needof such treatment an effective amount of a purified antagonist to RCN δ.

[0037] The invention also provides a method for detecting apolynucleotide which encodes RCN δ in a biological sample comprising thesteps of: a) hybridizing the complement of the polynucleotide sequencewhich encodes SEQ ID NO:3 to nucleic acid material of a biologicalsample, thereby forming a hybridization complex; and b) detecting thehybridization complex, wherein the presence of the complex correlateswith the presence of a polynucleotide encoding RCN δ in the biologicalsample. In one aspect the nucleic acid material of the biological sampleis amplified by the polymerase chain reaction prior to hybridization.

BRIEF DESCRIPTION OF THE FIGURES

[0038]FIGS. 1A, 1B, 1C, and 1D show the amino acid sequence (SEQ IDNO:1) and nucleic acid sequence (SEQ ID NO:2) of RCN γ. The alignmentwas produced using MacDNASIS PRO software (Hitachi Software EngineeringCo. Ltd. San Bruno, Calif.).

[0039]FIGS. 2A, 2B, 2C, 2D, 2E, 2F, and 2G show the amino acid sequence(SEQ ID NO:3) and nucleic acid sequence (SEQ ID NO:4) of RCN δ. Thealignment was produced using MacDNASIS PRO software (Hitachi SoftwareEngineering Co. Ltd. San Bruno, Calif.).

[0040]FIGS. 3A and 3B show the amino acid sequence alignments betweenRCN γ (922578; SEQ ID NO:1), human reticulocalbin (GI 1262329; SEQ IDNO:5), and rat brain clone REM#1 (GI 780361; SEQ ID NO:6), producedusing the multisequence alignment program of DNASTAR software (DNASTARInc., Madison Wis.).

[0041]FIGS. 4A and 4B show the amino acid sequence alignments betweenRCN δ (1601793; SEQ ID NO:3) and human reticulocalbin (GI 1262329; SEQID NO:5), produced using the multisequence alignment program of DNASTARsoftware (DNASTAR Inc., Madison Wis.).

[0042]FIGS. 5A, 5B, and 5C show the hydrophobicity plots for RCN γ (SEQID NO:1), RCN δ (SEQ ID NO:3), and human reticulocalbin (SEQ ID NO:5),respectively. The positive X axis reflects amino acid position, and thenegative Y axis, hydrophobicity (MACDNASIS PRO software).

DESCRIPTION OF THE INVENTION

[0043] Before the present proteins, nucleotide sequences, and methodsare described, it is understood that this invention is not limited tothe particular methodology, protocols, cell lines, vectors, and reagentsdescribed, as these may vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to limit the scope of the presentinvention which will be limited only by the appended claims.

[0044] It must be noted that as used herein and in the appended claims,the singular forms “a”, “an”, and “the” include plural reference unlessthe context clearly dictates otherwise. Thus, for example, reference to“a host cell” includes a plurality of such host cells, reference to the“antibody” is a reference to one or more antibodies and equivalentsthereof known to those skilled in the art, and so forth.

[0045] Unless defined otherwise, all technical and scientific terms usedherein have the same meanings as commonly understood by one of ordinaryskill in the art to which this invention belongs. Although any methodsand materials similar or equivalent to those described herein can beused in the practice or testing of the present invention, the preferredmethods, devices, and materials are now described. All publicationsmentioned herein are incorporated herein by reference for the purpose ofdescribing and disclosing the cell lines, vectors, and methodologieswhich are reported in the publications which might be used in connectionwith the invention. Nothing herein is to be construed as an admissionthat the invention is not entitled to antedate such disclosure by virtueof prior invention.

[0046] Definitions

[0047] RCN, as used herein, refers to the amino acid sequences ofsubstantially purified RCN obtained from any species, particularlymammalian, including bovine, ovine, porcine, murine, equine, andpreferably human, from any source whether natural, synthetic,semi-synthetic, or recombinant.

[0048] The term “agonist”, as used herein, refers to a molecule which,when bound to RCN, increases or prolongs the duration of the effect ofRCN. Agonists may include proteins, nucleic acids, carbohydrates, or anyother molecules which bind to and modulate the effect of RCN.

[0049] An “allele” or “allelic sequence”, as used herein, is analternative form of the gene encoding RCN. Alleles may result from atleast one mutation in the nucleic acid sequence and may result inaltered mRNAs or polypeptides whose structure or function may or may notbe altered. Any given natural or recombinant gene may have none, one, ormany allelic forms. Common mutational changes which give rise to allelesare generally ascribed to natural deletions, additions, or substitutionsof nucleotides. Each of these types of changes may occur alone, or incombination with the others, one or more times in a given sequence.

[0050] “Altered” nucleic acid sequences encoding RCN as used hereininclude those with deletions, insertions, or substitutions of differentnucleotides resulting in a polynucleotide that encodes the same or afunctionally equivalent RCN. Included within this definition arepolymorphisms which may or may not be readily detectable using aparticular oligonucleotide probe of the polynucleotide encoding RCN, andimproper or unexpected hybridization to alleles, with a locus other thanthe normal chromosomal locus for the polynucleotide sequence encodingRCN. The encoded protein may also be “altered” and contain deletions,insertions, or substitutions of amino acid residues which produce asilent change and result in a functionally equivalent RCN. Deliberateamino acid substitutions may be made on the basis of similarity inpolarity, charge, solubility, hydrophobicity, hydrophilicity, and/or theamphipathic nature of the residues as long as the biological orimmunological activity of RCN is retained. For example, negativelycharged amino acids may include aspartic acid and glutamic acid;positively charged amino acids may include lysine and arginine; andamino acids with uncharged polar head groups having similarhydrophilicity values may include leucine, isoleucine, and valine,glycine and alanine, asparagine and glutamine, serine and threonine, andphenylalanine and tyrosine.

[0051] “Amino acid sequence” as used herein refers to an oligopeptide,peptide, polypeptide, or protein sequence, and fragment thereof, and tonaturally occurring or synthetic molecules. Fragments of RCN arepreferably about 5 to about 15 amino acids in length and retain thebiological activity or the immunological activity of RCN. Where “aminoacid sequence” is recited herein to refer to an amino acid sequence of anaturally occurring protein molecule, amino acid sequence, and liketerms, are not meant to limit the amino acid sequence to the complete,native amino acid sequence associated with the recited protein molecule.

[0052] “Amplification” as used herein refers to the production ofadditional copies of a nucleic acid sequence and is generally carriedout using polymerase chain reaction (PCR) technologies well known in theart (Dieffenbach, C. W. and G. S. Dveksler (1995) PCR Primer, aLaboratory Manual, Cold Spring Harbor Press, Plainview, N.Y.).

[0053] The term “antagonist” as used herein, refers to a molecule which,when bound to RCN, decreases the amount or the duration of the effect ofthe biological or immunological activity of RCN. Antagonists may includeproteins, nucleic acids, carbohydrates, antibodies or any othermolecules which decrease the effect of RCN.

[0054] As used herein, the term “antibody” refers to intact molecules aswell as fragments thereof, such as Fab, F(ab′)₂, and Fv, which arecapable of binding the epitopic determinant. Antibodies that bind RCNpolypeptides can be prepared using intact polypeptides or fragmentscontaining small peptides of interest as the immunizing antigen. Thepolypeptide or oligopeptide used to immunize an animal can be derivedfrom the translation of RNA or synthesized chemically and can beconjugated to a carrier protein, if desired. Commonly used carriers thatare chemically coupled to peptides include bovine serum albumin andthyroglobulin, keyhole limpet hemocyanin. The coupled peptide is thenused to immunize the animal (e.g., a mouse, a rat, or a rabbit).

[0055] The term “antigenic determinant”, as used herein, refers to thatfragment of a molecule (i.e., an epitope) that makes contact with aparticular antibody. When a protein or fragment of a protein is used toimmunize a host animal, numerous regions of the protein may induce theproduction of antibodies which bind specifically to a given region orthree-dimensional structure on the protein; these regions or structuresare referred to as antigenic determinants. An antigenic determinant maycompete with the intact antigen (i.e., the immunogen used to elicit theimmune response) for binding to an antibody.

[0056] The term “antisense”, as used herein, refers to any compositioncontaining nucleotide sequences which are complementary to a specificDNA or RNA sequence. The term “antisense strand” is used in reference toa nucleic acid strand that is complementary to the “sense” strand.Antisense molecules include peptide nucleic acids and may be produced byany method including synthesis or transcription. Once introduced into acell, the complementary nucleotides combine with natural sequencesproduced by the cell to form duplexes and block either transcription ortranslation. The designation “negative” is sometimes used in referenceto the antisense strand, and “positive” is sometimes used in referenceto the sense strand.

[0057] The term “biologically active”, as used herein, refers to aprotein having structural, regulatory, or biochemical functions of anaturally occurring molecule. Likewise, “immunologically active” refersto the capability of the natural, recombinant, or synthetic RCN, or anyoligopeptide thereof, to induce a specific immune response inappropriate animals or cells and to bind with specific antibodies.

[0058] The terms “complementary” or “complementarity”, as used herein,refer to the natural binding of polynucleotides under permissive saltand temperature conditions by base-pairing. For example, the sequence“A-G-T” binds to the complementary sequence “T-C-A”. Complementaritybetween two single-stranded molecules may be “partial”, in which onlysome of the nucleic acids bind, or it may be complete when totalcomplementarity exists between the single stranded molecules. The degreeof complementarity between nucleic acid strands has significant effectson the efficiency and strength of hybridization between nucleic acidstrands. This is of particular importance in amplification reactions,which depend upon binding between nucleic acids strands and in thedesign and use of PNA molecules.

[0059] A “composition comprising a given polynucleotide sequence” asused herein refers broadly to any composition containing the givenpolynucleotide sequence. The composition may comprise a dry formulationor an aqueous solution. Compositions comprising polynucleotide sequencesencoding RCN (SEQ ID NO:1, SEQ ID NO:3) or fragments thereof (e.g., SEQID NO:2 or SEQ ID NO:4 and fragments thereof) may be employed ashybridization probes. The probes may be stored in freeze-dried form andmay be associated with a stabilizing agent such as a carbohydrate. Inhybridizations, the probe may be deployed in an aqueous solutioncontaining salts (e.g., NaCl), detergents (e.g., SDS) and othercomponents (e.g., Denhardt's solution, dry milk, salmon sperm DNA,etc.).

[0060] “Consensus”, as used herein, refers to a nucleic acid sequencewhich has been resequenced to resolve uncalled bases, has been extendedusing XL-PCR (Perkin Elmer, Norwalk, Conn.) in the 5′ and/or the 3′direction and resequenced, or has been assembled from the overlappingsequences of more than one Incyte Clone using a computer program forfragment assembly (e.g., GELVIEW Fragment Assembly system, GCG, Madison,Wis.). Some sequences have been both extended and assembled to producethe consensus sequence.

[0061] The term “correlates with expression of a polynucleotide”, asused herein, indicates that the detection of the presence of ribonucleicacid that is similar to SEQ ID NO:2, or SEQ ID NO:4 by northern analysisis indicative of the presence of mRNA encoding RCN in a sample andthereby correlates with expression of the transcript from thepolynucleotide encoding the protein.

[0062] A “deletion”, as used herein, refers to a change in the aminoacid or nucleotide sequence and results in the absence of one or moreamino acid residues or nucleotides.

[0063] The term “derivative”, as used herein, refers to the chemicalmodification of a nucleic acid encoding or complementary to RCN or theencoded RCN. Such modifications include, for example, replacement ofhydrogen by an alkyl, acyl, or amino group. A nucleic acid derivativeencodes a polypeptide which retains the biological or immunologicalfunction of the natural molecule. A derivative polypeptide is one whichis modified by glycosylation, pegylation, or any similar process whichretains the biological or immunological function of the polypeptide fromwhich it was derived.

[0064] The term “homology”, as used herein, refers to a degree ofcomplementarity. There may be partial homology or complete homology(i.e., identity). A partially complementary sequence that at leastpartially inhibits an identical sequence from hybridizing to a targetnucleic acid is referred to using the functional term “substantiallyhomologous.” The inhibition of hybridization of the completelycomplementary sequence to the target sequence may be examined using ahybridization assay (Southern or northern blot, solution hybridizationand the like) under conditions of low stringency. A substantiallyhomologous sequence or hybridization probe will compete for and inhibitthe binding of a completely homologous sequence to the target sequenceunder conditions of low stringency. This is not to say that conditionsof low stringency are such that non-specific binding is permitted; lowstringency conditions require that the binding of two sequences to oneanother be a specific (i.e., selective) interaction. The absence ofnon-specific binding may be tested by the use of a second targetsequence which lacks even a partial degree of complementarity (e.g.,less than about 30% identity). In the absence of non-specific binding,the probe will not hybridize to the second non-complementary targetsequence.

[0065] Human artificial chromosomes (HACs) are linear microchromosomeswhich may contain DNA sequences of 1K to 10M in size and contain all ofthe elements required for stable mitotic chromosome segregation andmaintenance (Harrington, J. J. et al. (1997) Nat Genet. 15:345-355).

[0066] The term “humanized antibody”, as used herein, refers to antibodymolecules in which amino acids have been replaced in the non-antigenbinding regions in order to more closely resemble a human antibody,while still retaining the original binding ability.

[0067] The term “hybridization”, as used herein, refers to any processby which a strand of nucleic acid binds with a complementary strandthrough base pairing.

[0068] The term “hybridization complex”, as used herein, refers to acomplex formed between two nucleic acid sequences by virtue of theformation of hydrogen bonds between complementary G and C bases andbetween complementary A and T bases; these hydrogen bonds may be furtherstabilized by base stacking interactions. The two complementary nucleicacid sequences hydrogen bond in an antiparallel configuration. Ahybridization complex may be formed in solution (e.g., C₀t or R₀tanalysis) or between one nucleic acid sequence present in solution andanother nucleic acid sequence immobilized on a solid support (e.g.,paper, membranes, filters, chips, pins or glass slides, or any otherappropriate substrate to which cells or their nucleic acids have beenfixed).

[0069] An “insertion” or “addition”, as used herein, refers to a changein an amino acid or nucleotide sequence resulting in the addition of oneor more amino acid residues or nucleotides, respectively, as compared tothe naturally occurring molecule.

[0070] “Microarray” refers to an array of distinct polynucleotides oroligonucleotides synthesized on a substrate, such as paper, nylon orother type of membrane, filter, chip, glass slide, or any other suitablesolid support.

[0071] The term “modulate”, as used herein, refers to a change in theactivity of RCN. For example, modulation may cause an increase or adecrease in protein activity, binding characteristics, or any otherbiological, functional or immunological properties of RCN.

[0072] “Nucleic acid sequence” as used herein refers to anoligonucleotide, nucleotide, or polynucleotide, and fragments thereof,and to DNA or RNA of genomic or synthetic origin which may be single- ordouble-stranded, and represent the sense or antisense strand.“Fragments” are those nucleic acid sequences which are greater than 60nucleotides than in length, and most preferably includes fragments thatare at least 100 nucleotides or at least 1000 nucleotides, and at least10,000 nucleotides in length.

[0073] The term “oligonucleotide” refers to a nucleic acid sequence ofat least about 6 nucleotides to about 60 nucleotides, preferably about15 to 30 nucleotides, and more preferably about 20 to 25 nucleotides,which can be used in PCR amplification or a hybridization assay, or amicroarray. As used herein, oligonucleotide is substantially equivalentto the terms “amplimers”, “primers”, “oligomers”, and “probes”, ascommonly defined in the art.

[0074] “Peptide nucleic acid”, PNA as used herein, refers to anantisense molecule or anti-gene agent which comprises an oligonucleotideof at least five nucleotides in length linked to a peptide backbone ofamino acid residues which ends in lysine. The terminal lysine conferssolubility to the composition. PNAs may be pegylated to extend theirlifespan in the cell where they preferentially bind complementary singlestranded DNA and RNA and stop transcript elongation (Nielsen, P. E. etal. (1993) Anticancer Drug Des. 8:53-63).

[0075] The term “portion”, as used herein, with regard to a protein (asin “a portion of a given protein”) refers to fragments of that protein.The fragments may range in size from five amino acid residues to theentire amino acid sequence minus one amino acid. Thus, a protein“comprising at least a portion of the amino acid sequence of SEQ IDNO:1” encompasses the full-length RCN γ and fragments thereof, and aprotein “comprising at least a portion of the amino acid sequence of SEQID NO:3” encompasses the full-length RCN δ and fragments thereof.

[0076] The term “sample”, as used herein, is used in its broadest sense.A biological sample suspected of containing nucleic acid encoding RCN,or fragments thereof, or RCN itself may comprise a bodily fluid, extractfrom a cell, chromosome, organelle, or membrane isolated from a cell, acell, genomic DNA, RNA, or cDNA(in solution or bound to a solid support,a tissue, a tissue print, and the like.

[0077] The terms “specific binding” or “specifically binding”, as usedherein, refers to that interaction between a protein or peptide and anagonist, an antibody and an antagonist. The interaction is dependentupon the presence of a particular structure (i.e., the antigenicdeterminant or epitope) of the protein recognized by the bindingmolecule. For example, if an antibody is specific for epitope “A”, thepresence of a protein containing epitope A (or free, unlabeled A) in areaction containing labeled “A” and the antibody will reduce the amountof labeled A bound to the antibody.

[0078] The terms “stringent conditions” or “stringency”, as used herein,refer to the conditions for hybridization as defined by the nucleicacid, salt, and temperature. These conditions are well known in the artand may be altered in order to identify or detect identical or relatedpolynucleotide sequences. Numerous equivalent conditions comprisingeither low or high stringency depend on factors such as the length andnature of the sequence (DNA, RNA, base composition), nature of thetarget (DNA, RNA, base composition), milieu (in solution or immobilizedon a solid substrate), concentration of salts and other components(e.g., formamide, dextran sulfate and/or polyethylene glycol), andtemperature of the reactions (within a range from about 5° C. below themelting temperature of the probe to about 20° C. to 25° C. below themelting temperature). One or more factors be may be varied to generateconditions of either low or high stringency different from, butequivalent to, the above listed conditions.

[0079] The term “substantially purified”, as used herein, refers tonucleic or amino acid sequences that are removed from their naturalenvironment, isolated or separated, and are at least 60% free,preferably 75% free, and most preferably 90% free from other componentswith which they are naturally associated.

[0080] A “substitution”, as used herein, refers to the replacement ofone or more amino acids or nucleotides by different amino acids ornucleotides, respectively.

[0081] “Transformation”, as defined herein, describes a process by whichexogenous DNA enters and changes a recipient cell. It may occur undernatural or artificial conditions using various methods well known in theart. Transformation may rely on any known method for the insertion offoreign nucleic acid sequences into a prokaryotic or eukaryotic hostcell. The method is selected based on the type of host cell beingtransformed and may include, but is not limited to, viral infection,electroporation, heat shock, lipofection, and particle bombardment. Such“transformed” cells include stably transformed cells in which theinserted DNA is capable of replication either as an autonomouslyreplicating plasmid or as part of the host chromosome. They also includecells which transiently express the inserted DNA or RNA for limitedperiods of time.

[0082] A “variant” of RCN, as used herein, refers to an amino acidsequence that is altered by one or more amino acids. The variant mayhave “conservative” changes, wherein a substituted amino acid hassimilar structural or chemical properties, e.g., replacement of leucinewith isoleucine. More rarely, a variant may have “nonconservative”changes, e.g., replacement of a glycine with a tryptophan. Analogousminor variations may also include amino acid deletions or insertions, orboth. Guidance in determining which amino acid residues may besubstituted, inserted, or deleted without abolishing biological orimmunological activity may be found using computer programs well knownin the art, for example, DNASTAR software.

[0083] The Invention

[0084] The invention is based on the discovery of two new humanreticulocalbin isoforms (collectively referred to as “RCN” andindividually, as “RCN γ” and “RCN δ”), the polynucleotides encoding RCN,and the use of these compositions for the diagnosis, prevention, ortreatment of infectious, developmental, neoplastic, and immunologicaldisorders.

[0085] Nucleic acids encoding the RCN γ of the present invention werefirst identified in Incyte Clone 922578 from the right atrium tissuecDNA library (RATRNOT02) using a computer search for amino acid sequencealignments. A consensus sequence, SEQ ID NO:2, was derived from thefollowing overlapping and/or extended nucleic acid sequences: IncyteClones 922578 (RATRNOT02), 1330666 (PANCNOT07), 1852086 (LUNGFET03),1878487 (LEUKNOT03), 2309791 (NGANNOT01), and 2539327 (BONRTUT01).

[0086] In one embodiment, the invention encompasses a polypeptidecomprising the amino acid sequence of SEQ ID NO:1, as shown in FIGS. 1A,1B, 1C, and 1D. RCN γ is 328 amino acids in length, has an N-terminalsignal peptide (M-1 to G-17), has six EF-hands, and has an HDELC-terminal tetrapeptide. Five of the EF-hands have a conserved glycineresidue in the central portion of the domain. RCN γ has one potentialN-glycosylation site at residue N-140; eight potential casein kinase IIphosphorylation sites at residues T-72, S-98, T-127, T-184, T-208,S-289, T-291, and S-298; and four potential protein kinase Cphosphorylation sites at residues T-127, T-160, T-244, and T-291. Asshown in FIGS. 3A and 3B, RCN γ has chemical and structural homologywith human reticulocalbin (GI 1262329; SEQ ID NO:5) and rat clone REM#1(GI 780361; SEQ ID NO:6). In particular, RCN γ and human reticulocalbinshare 52% amino acid residue identity over the full length of RCN γ. RCNγ and human reticulocalbin share an N-terminal signal peptide, six ofthe EF-hands, and an HDEL C-terminal tetrapeptide. RCN γ and rat cloneREM#1 share 91% amino acid residue identity over the length of REM#1.RCN γ amino acid residues G-61 to A-158 are homologous to REM#1, and RCNγ shares one EF-hand with REM#1.

[0087] As illustrated by FIGS. 5A and 5C, RCN γ and human reticulocalbinhave rather similar hydrophobicity plots. Northern analysis shows theexpression of this sequence in various libraries, at least 44% of whichare immortalized or cancerous, at least 17% of which involve immuneresponse, and at least 22% of which involve fetal or rapidly dividingtissue. Of particular note is the expression of RCN γ in lung, brain,breast, smooth muscle, and endothelial cells, and at sites ofhematopoiesis.

[0088] Nucleic acids encoding the RCN δ of the present invention werefirst identified in Incyte Clone 1601793 from the bladder cDNA library(BLADNOT03) using a computer search for amino acid sequence alignments.A consensus sequence, SEQ ID NO:4, was derived from the followingoverlapping and/or extended nucleic acid sequences: Incyte Clones1601793 (BLADNOT03), 1273785 (TESTTUT02), 1404618 (LATRTUT02), 1691170(PROSTUT10), 980872 (TONGTUT01), 2344906 (TESTTUT02), 2174719(ENDCNOT03), and 1820444 (GBLATUT01).

[0089] In one embodiment, the invention encompasses a polypeptidecomprising the amino acid sequence of SEQ ID NO:3, as shown in FIGS. 2A,2B, 2C, 2D, 2E, 2F, and 2G. RCN δ is 315 amino acids in length, has anN-terminal signal peptide (M-1 to S-19), and has seven EF-hands. Allseven of the EF-hands have a conserved glycine residue in the centralportion of the domain. RCN δ has one potential N-glycosylation site atN-131; nine potential casein kinase II phosphorylation sites at residuesT-65, S-78, T-89, S-125, T-172, T-196, T-232, S-261, and T-286; andthree potential protein kinase C phosphorylation sites at residues T-22,S-35, and T-232. As shown in FIGS. 4A and 4B, RCN δ has chemical andstructural homology with human reticulocalbin (GI 1262329; SEQ ID NO:5).In particular, RCN δ and human reticulocalbin share 58% identity, sharean N-terminal signal peptide, and six EF-hands. As illustrated by FIGS.5B and 5C, RCN δ and human reticulocalbin have rather similarhydrophobicity plots. Northern analysis shows the expression of thissequence in various libraries, at least 50% of which are immortalized orcancerous, at least 22% of which involve immune response, and at least19% of which involve fetal or proliferating tissue. Of particular noteis the expression of RCN δ in heart, gut, prostate, and smooth muscle;and at sites of hematopoiesis.

[0090] The invention also encompasses RCN variants. A preferred RCNvariant is one having at least 80%, and more preferably at least 90%,amino acid sequence identity to the RCN amino acid sequence (SEQ IDNO:1, or SEQ ID NO:3) and which retains at least one biological,immunological or other functional characteristic or activity of RCN. Amost preferred RCN variant is one having at least 95% amino acidsequence identity to SEQ ID NO:1, or SEQ ID NO:3.

[0091] The invention also encompasses polynucleotides which encode RCN.Accordingly, any nucleic acid sequence which encodes the amino acidsequence of RCN can be used to produce recombinant molecules whichexpress RCN. In a particular embodiment, the invention encompasses thepolynucleotide comprising the nucleic acid sequence of SEQ ID NO:2 asshown in FIGS. 1A, 1B, 1C, and 1D. In another embodiment, the inventionencompasses the polynucleotide comprising the nucleic acid sequence ofSEQ ID NO:4 as shown in FIGS. 2A, 2B, 2C, 2D, 2E, 2F, and 2G.

[0092] It will be appreciated by those skilled in the art that as aresult of the degeneracy of the genetic code, a multitude of nucleotidesequences encoding RCN, some bearing minimal homology to the nucleotidesequences of any known and naturally occurring gene, may be produced.Thus, the invention contemplates each and every possible variation ofnucleotide sequence that could be made by selecting combinations basedon possible codon choices. These combinations are made in accordancewith the standard triplet genetic code as applied to the nucleotidesequence of naturally occurring RCN, and all such variations are to beconsidered as being specifically disclosed.

[0093] Although nucleotide sequences which encode RCN and its variantsare preferably capable of hybridizing to the nucleotide sequence of thenaturally occurring RCN under appropriately selected conditions ofstringency, it may be advantageous to produce nucleotide sequencesencoding RCN or its derivatives possessing a substantially differentcodon usage. Codons may be selected to increase the rate at whichexpression of the peptide occurs in a particular prokaryotic oreukaryotic host in accordance with the frequency with which particularcodons are utilized by the host. Other reasons for substantiallyaltering the nucleotide sequence encoding RCN and its derivativeswithout altering the encoded amino acid sequences include the productionof RNA transcripts having more desirable properties, such as a greaterhalf-life, than transcripts produced from the naturally occurringsequence.

[0094] The invention also encompasses production of DNA sequences, orfragments thereof, which encode RCN and its derivatives, entirely bysynthetic chemistry. After production, the synthetic sequence may beinserted into any of the many available expression vectors and cellsystems using reagents that are well known in the art. Moreover,synthetic chemistry may be used to introduce mutations into a sequenceencoding RCN or any fragment thereof.

[0095] Also encompassed by the invention are polynucleotide sequencesthat are capable of hybridizing to the claimed nucleotide sequences, andin particular, those shown in SEQ ID NO:2 or SEQ ID NO:4, under variousconditions of stringency as taught in Wahl, G. M. and S. L. Berger(1987; Methods Enzymol. 152:399-407) and Kimmel, A. R. (1987; MethodsEnzymol. 152:507-511).

[0096] Methods for DNA sequencing which are well known and generallyavailable in the art and may be used to practice any of the embodimentsof the invention. The methods may employ such enzymes as the Klenowfragment of DNA polymerase I, Sequenase (U.S. Biochemical Corp,Cleveland, Ohio), Taq polymerase (Perkin Elmer), thermostable T7polymerase (Amersham, Chicago, Ill.), or combinations of polymerases andproofreading exonucleases such as those found in the ELONGASEamplification system marketed by Gibco/BRL (Gaithersburg, Md.).Preferably, the process is automated with machines such as the MICROLAB2200 (Hamilton, Reno, Nev.), Peltier thermal cycler (PTC200; M JResearch, Watertown, Mass.), and the ABI Catalyst and 373 and 377 DNAsequencers (Perkin Elmer).

[0097] The nucleic acid sequences encoding RCN may be extended utilizinga partial nucleotide sequence and employing various methods known in theart to detect upstream sequences such as promoters and regulatoryelements. For example, one method which may be employed,“restriction-site” PCR, uses universal primers to retrieve unknownsequence adjacent to a known locus (Sarkar, G. (1993) PCR MethodsApplic. 2:318-322). In particular, genomic DNA is first amplified in thepresence of primer to a linker sequence and a primer specific to theknown region. The amplified sequences are then subjected to a secondround of PCR with the same linker primer and another specific primerinternal to the first one. Products of each round of PCR are transcribedwith an appropriate RNA polymerase and sequenced using reversetranscriptase.

[0098] Inverse PCR may also be used to amplify or extend sequences usingdivergent primers based on a known region (Triglia, T. et al. (1988)Nucleic Acids Res. 16:8186). The primers may be designed usingcommercially available software such as OLIGO 4.06 primer analysissoftware (National Biosciences Inc., Plymouth, Min.), or anotherappropriate program, to be 22-30 nucleotides in length, to have a GCcontent of 50% or more, and to anneal to the target sequence attemperatures about 68°-72° C. The method uses several restrictionenzymes to generate a suitable fragment in the known region of a gene.The fragment is then circularized by intramolecular ligation and used asa PCR template.

[0099] Another method which may be used is capture PCR which involvesPCR amplification of DNA fragments adjacent to a known sequence in humanand yeast artificial chromosome DNA (Lagerstrom, M. et al. (1991) PCRMethods Applic. 1:111-119). In this method, multiple restriction enzymedigestions and ligations may also be used to place an engineereddouble-stranded sequence into an unknown fragment of the DNA moleculebefore performing PCR.

[0100] Another method which may be used to retrieve unknown sequences isthat of Parker, J. D. et al. (1991; Nucleic Acids Res. 19:3055-3060).Additionally, one may use PCR, nested primers, and PROMOTERFINDERlibraries to walk genomic DNA (Clontech, Palo Alto, Calif.). Thisprocess avoids the need to screen libraries and is useful in findingintron/exon junctions. When screening for full-length cDNAs, it ispreferable to use libraries that have been size-selected to includelarger cDNAs. Also, random-primed libraries are preferable, in that theywill contain more sequences which contain the 5′ regions of genes. Useof a randomly primed library may be especially preferable for situationsin which an oligo d(T) library does not yield a full-length cDNA.Genomic libraries may be useful for extension of sequence into 5′non-transcribed regulatory regions.

[0101] Capillary electrophoresis systems which are commerciallyavailable may be used to analyze the size or confirm the nucleotidesequence of sequencing or PCR products. In particular, capillarysequencing may employ flowable polymers for electrophoretic separation,four different fluorescent dyes (one for each nucleotide) which arelaser activated, and detection of the emitted wavelengths by a chargecoupled devise camera. Output/light intensity may be converted toelectrical signal using appropriate software (e.g. GENOTYPER andSEQUENCE NAVIGATOR, Perkin Elmer) and the entire process from loading ofsamples to computer analysis and electronic data display may be computercontrolled. Capillary electrophoresis is especially preferable for thesequencing of small pieces of DNA which might be present in limitedamounts in a particular sample.

[0102] In another embodiment of the invention, polynucleotide sequencesor fragments thereof which encode RCN may be used in recombinant DNAmolecules to direct expression of RCN, fragments or functionalequivalents thereof, in appropriate host cells. Due to the inherentdegeneracy of the genetic code, other DNA sequences which encodesubstantially the same or a functionally equivalent amino acid sequencemay be produced, and these sequences may be used to clone and expressRCN.

[0103] As will be understood by those of skill in the art, it may beadvantageous to produce RCN-encoding nucleotide sequences possessingnon-naturally occurring codons. For example, codons preferred by aparticular prokaryotic or eukaryotic host can be selected to increasethe rate of protein expression or to produce an RNA transcript havingdesirable properties, such as a half-life which is longer than that of atranscript generated from the naturally occurring sequence.

[0104] The nucleotide sequences of the present invention can beengineered using methods generally known in the art in order to alterRCN encoding sequences for a variety of reasons, including but notlimited to, alterations which modify the cloning, processing, and/orexpression of the gene product. DNA shuffling by random fragmentationand PCR reassembly of gene fragments and synthetic oligonucleotides maybe used to engineer the nucleotide sequences. For example, site-directedmutagenesis may be used to insert new restriction sites, alterglycosylation patterns, change codon preference, produce splicevariants, introduce mutations, and so forth.

[0105] In another embodiment of the invention, natural, modified, orrecombinant nucleic acid sequences encoding RCN may be ligated to aheterologous sequence to encode a fusion protein. For example, to screenpeptide libraries for inhibitors of RCN activity, it may be useful toencode a chimeric RCN protein that can be recognized by a commerciallyavailable antibody. A fusion protein may also be engineered to contain acleavage site located between the RCN encoding sequence and theheterologous protein sequence, so that RCN may be cleaved and purifiedaway from the heterologous moiety.

[0106] In another embodiment, sequences encoding RCN may be synthesized,in whole or in part, using chemical methods well known in the art (seeCaruthers, M. H. et al. (1980) Nucl. Acids Res. Symp. Ser. 215-223,Horn, T. et al. (1980) Nucl. Acids Res. Symp. Ser. 225-232).Alternatively, the protein itself may be produced using chemical methodsto synthesize the amino acid sequence of RCN, or a fragment thereof. Forexample, peptide synthesis can be performed using various solid-phasetechniques (Roberge, J. Y. et al. (1995) Science 269:202-204) andautomated synthesis may be achieved, for example, using the ABI 431Apeptide synthesizer (Perkin Elmer).

[0107] The newly synthesized peptide may be substantially purified bypreparative high performance liquid chromatography (e.g., Creighton, T.(1983) Proteins, Structures and Molecular Principles, W H Freeman andCo., New York, N.Y.). The composition of the synthetic peptides may beconfirmed by amino acid analysis or sequencing (e.g., the Edmandegradation procedure; Creighton, supra). Additionally, the amino acidsequence of RCN, or any part thereof, may be altered during directsynthesis and/or combined using chemical methods with sequences fromother proteins, or any part thereof, to produce a variant polypeptide.

[0108] In order to express a biologically active RCN, the nucleotidesequences encoding RCN or functional equivalents, may be inserted intoappropriate expression vector, i.e., a vector which contains thenecessary elements for the transcription and translation of the insertedcoding sequence.

[0109] Methods which are well known to those skilled in the art may beused to construct expression vectors containing sequences encoding RCNand appropriate transcriptional and translational control elements.These methods include in vitro recombinant DNA techniques, synthetictechniques, and in vivo genetic recombination. Such techniques aredescribed in Sambrook, J. et al. (1989) Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Press, Plainview, N.Y., and Ausubel, F. M. etal. (1989) Current Protocols in Molecular Biology, John Wiley & Sons,New York, N.Y.

[0110] A variety of expression vector/host systems may be utilized tocontain and express sequences encoding RCN. These include, but are notlimited to, microorganisms such as bacteria transformed with recombinantbacteriophage, plasmid, or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors; insect cell systems infectedwith virus expression vectors (e.g., baculovirus); plant cell systemstransformed with virus expression vectors (e.g., cauliflower mosaicvirus, CaMV; tobacco mosaic virus, TMV) or with bacterial expressionvectors (e.g., Ti or pBR322 plasmids); or animal cell systems. Theinvention is not limited by the host cell employed.

[0111] The “control elements” or “regulatory sequences” are thosenon-translated regions of the vector—enhancers, promoters, 5′ and 3′untranslated regions—which interact with host cellular proteins to carryout transcription and translation. Such elements may vary in theirstrength and specificity. Depending on the vector system and hostutilized, any number of suitable transcription and translation elements,including constitutive and inducible promoters, may be used. Forexample, when cloning in bacterial systems, inducible promoters such asthe hybrid lacZ promoter of the BLUESCRIPT phagemid (Stratagene,LaJolla, Calif.) or PSPORT1 plasmid (Gibco BRL) and the like may beused. The baculovirus polyhedrin promoter may be used in insect cells.Promoters or enhancers derived from the genomes of plant cells (e.g.,heat shock, RUBISCO; and storage protein genes) or from plant viruses(e.g., viral promoters or leader sequences) may be cloned into thevector. In mammalian cell systems, promoters from mammalian genes orfrom mammalian viruses are preferable. If it is necessary to generate acell line that contains multiple copies of the sequence encoding RCN,vectors based on SV40 or EBV may be used with an appropriate selectablemarker.

[0112] In bacterial systems, a number of expression vectors may beselected depending upon the use intended for RCN. For example, whenlarge quantities of RCN are needed for the induction of antibodies,vectors which direct high level expression of fusion proteins that arereadily purified may be used. Such vectors include, but are not limitedto, the multifunctional E. coli cloning and expression vectors such asBLUESCRIPT (Stratagene), in which the sequence encoding RCN may beligated into the vector in frame with sequences for the amino-terminalMet and the subsequent 7 residues of β-galactosidase so that a hybridprotein is produced; pIN vectors (Van Heeke, G. and S. M. Schuster(1989) J. Biol. Chem. 264:5503-5509); and the like. pGEX vectors(Promega, Madison, Wis.) may also be used to express foreignpolypeptides as fusion proteins with glutathione S-transferase (GST). Ingeneral, such fusion proteins are soluble and can easily be purifiedfrom lysed cells by adsorption to glutathione-agarose beads followed byelution in the presence of free glutathione. Proteins made in suchsystems may be designed to include heparin, thrombin, or factor XAprotease cleavage sites so that the cloned polypeptide of interest canbe released from the GST moiety at will.

[0113] In the yeast, Saccharomyces cerevisiae, a number of vectorscontaining constitutive or inducible promoters such as alpha factor,alcohol oxidase, and PGH may be used. For reviews, see Ausubel et al.(supra) and Grant et al. (1987) Methods Enzymol. 153:516-544.

[0114] In cases where plant expression vectors are used, the expressionof sequences encoding RCN may be driven by any of a number of promoters.For example, viral promoters such as the 35S and 19S promoters of CaMVmay be used alone or in combination with the omega leader sequence fromTMV (Takamatsu, N. (1987) EMBO J. 6:307-311). Alternatively, plantpromoters such as the small subunit of RUBISCO or heat shock promotersmay be used (Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680; Broglie, R.et al. (1984) Science 224:838-843; and Winter, J. et al. (1991) ResultsProbl. Cell Differ. 17:85-105). These constructs can be introduced intoplant cells by direct DNA transformation or pathogen-mediatedtransfection. Such techniques are described in a number of generallyavailable reviews (see, for example, Hobbs, S. or Murry, L. E. in McGrawHill Yearbook of Science and Technology (1992) McGraw Hill, New York,N.Y.; pp. 191-196.

[0115] An insect system may also be used to express RCN. For example, inone such system, Autographa californica nuclear polyhedrosis virus(AcNPV) is used as a vector to express foreign genes in Spodopterafrugiperda cells or in Trichoplusia larvae. The sequences encoding RCNmay be cloned into a non-essential region of the virus, such as thepolyhedrin gene, and placed under control of the polyhedrin promoter.Successful insertion of RCN will render the polyhedrin gene inactive andproduce recombinant virus lacking coat protein. The recombinant virusesmay then be used to infect, for example, S. frugiperda cells orTrichoplusia larvae in which RCN may be expressed (Engelhard, E. K. etal. (1994) Proc. Nat. Acad. Sci. 91:3224-3227).

[0116] In mammalian host cells, a number of viral-based expressionsystems may be utilized. In cases where an adenovirus is used as anexpression vector, sequences encoding RCN may be ligated into anadenovirus transcription/translation complex consisting of the latepromoter and tripartite leader sequence. Insertion in a non-essential E1or E3 region of the viral genome may be used to obtain a viable viruswhich is capable of expressing RCN in infected host cells (Logan, J. andShenk, T. (1984) Proc. Natl. Acad. Sci. 81:3655-3659). In addition,transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer,may be used to increase expression in mammalian host cells.

[0117] Human artificial chromosomes (HACs) may also be employed todeliver larger fragments of DNA than can be contained and expressed in aplasmid. HACs of 6 to 10M are constructed and delivered via conventionaldelivery methods (liposomes, polycationic amino polymers, or vesicles)for therapeutic purposes.

[0118] Specific initiation signals may also be used to achieve moreefficient translation of sequences encoding RCN. Such signals includethe ATG initiation codon and adjacent sequences. In cases wheresequences encoding RCN, its initiation codon, and upstream sequences areinserted into the appropriate expression vector, no additionaltranscriptional or translational control signals may be needed. However,in cases where only coding sequence, or a fragment thereof, is inserted,exogenous translational control signals including the ATG initiationcodon should be provided. Furthermore, the initiation codon should be inthe correct reading frame to ensure translation of the entire insert.Exogenous translational elements and initiation codons may be of variousorigins, both natural and synthetic. The efficiency of expression may beenhanced by the inclusion of enhancers which are appropriate for theparticular cell system which is used, such as those described in theliterature (Scharf, D. et al. (1994) Results Probl. Cell Differ.20:125-162).

[0119] In addition, a host cell strain may be chosen for its ability tomodulate the expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation, and acylation.Post-translational processing which cleaves a “prepro” form of theprotein may also be used to facilitate correct insertion, folding and/orfunction. Different host cells which have specific cellular machineryand characteristic mechanisms for post-translational activities (e.g.,CHO, HeLa, MDCK, HEK293, and W138), are available from the American TypeCulture Collection (ATCC; Bethesda, Md.) and may be chosen to ensure thecorrect modification and processing of the foreign protein.

[0120] For long-term, high-yield production of recombinant proteins,stable expression is preferred. For example, cell lines which stablyexpress RCN may be transformed using expression vectors which maycontain viral origins of replication and/or endogenous expressionelements and a selectable marker gene on the same or on a separatevector. Following the introduction of the vector, cells may be allowedto grow for 1-2 days in an enriched media before they are switched toselective media. The purpose of the selectable marker is to conferresistance to selection, and its presence allows growth and recovery ofcells which successfully express the introduced sequences. Resistantclones of stably transformed cells may be proliferated using tissueculture techniques appropriate to the cell type.

[0121] Any number of selection systems may be used to recovertransformed cell lines. These include, but are not limited to, theherpes simplex virus thymidine kinase (Wigler, M. et al. (1977) Cell11:223-32) and adenine phosphoribosyltransferase (Lowy, I. et al. (1980)Cell 22:817-23) genes which can be employed in tk- or aprt-cells,respectively. Also, antimetabolite, antibiotic, or herbicide resistancecan be used as the basis for selection: for example, dhfr which confersresistance to methotrexate (Wigler, M. et al. (1980) Proc. Natl. Acad.Sci. 77:3567-70); npt, which confers resistance to the aminoglycosidesneomycin and G-418 (Colbere-Garapin, F. et al (1981) J. Mol. Biol.150:1-14), and als or pat, which confer resistance to chlorsulfuron andphosphinotricin acetyltransferase, respectively (Murry, supra).Additional selectable genes have been described, for example, trpB,which allows cells to utilize indole in place of tryptophan, or hisD,which allows cells to utilize histinol in place of histidine (Hartman,S. C. and R. C. Mulligan (1988) Proc. Natl. Acad. Sci. 85:8047-51).Recently, the use of visible markers has gained popularity with suchmarkers as anthocyanins, β glucuronidase and its substrate GUS, andluciferase and its substrate luciferin, being widely used not only toidentify transformants, but also to quantify the amount of transient orstable protein expression attributable to a specific vector system(Rhodes, C. A. et al. (1995) Methods Mol. Biol. 55:121-131).

[0122] Although the presence/absence of marker gene expression suggeststhat the gene of interest is also present, its presence and expressionmay need to be confirmed. For example, if the sequence encoding RCN isinserted within a marker gene sequence, transformed cells containingsequences encoding RCN can be identified by the absence of marker genefunction. Alternatively, a marker gene can be placed in tandem with asequence encoding RCN under the control of a single promoter. Expressionof the marker gene in response to induction or selection usuallyindicates expression of the tandem gene as well.

[0123] Alternatively, host cells which contain the nucleic acid sequenceencoding RCN and express RCN may be identified by a variety ofprocedures known to those of skill in the art. These procedures include,but are not limited to, DNA-DNA or DNA-RNA hybridizations and proteinbioassay or immunoassay techniques which include membrane, solution, orchip based technologies for the detection and/or quantification ofnucleic acid or protein.

[0124] The presence of polynucleotide sequences encoding RCN can bedetected by DNA-DNA or DNA-RNA hybridization or amplification usingprobes or fragments or fragments of polynucleotides encoding RCN.Nucleic acid amplification based assays involve the use ofoligonucleotides or oligomers based on the sequences encoding RCN todetect transformants containing DNA or RNA encoding RCN.

[0125] A variety of protocols for detecting and measuring the expressionof RCN, using either polyclonal or monoclonal antibodies specific forthe protein are known in the art. Examples include enzyme-linkedimmunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescenceactivated cell sorting (FACS). A two-site, monoclonal-based immunoassayutilizing monoclonal antibodies reactive to two non-interfering epitopeson RCN is preferred, but a competitive binding assay may be employed.These and other assays are described, among other places, in Hampton, R.et al. (1990; Serological Methods, a Laboratory Manual, APS Press, StPaul, Min.) and Maddox, D. E. et al. (1983; J. Exp. Med. 158:1211-1216).

[0126] A wide variety of labels and conjugation techniques are known bythose skilled in the art and may be used in various nucleic acid andamino acid assays. Means for producing labeled hybridization or PCRprobes for detecting sequences related to polynucleotides encoding RCNinclude oligolabeling, nick translation, end-labeling or PCRamplification using a labeled nucleotide. Alternatively, the sequencesencoding RCN, or any fragments thereof may be cloned into a vector forthe production of an mRNA probe. Such vectors are known in the art, arecommercially available, and may be used to synthesize RNA probes invitro by addition of an appropriate RNA polymerase such as T7, T3, orSP6 and labeled nucleotides. These procedures may be conducted using avariety of commercially available kits (Pharmacia & Upjohn (Kalamazoo,Mich.); Promega (Madison Wis.); and U.S. Biochemical Corp., Cleveland,Ohio)). Suitable reporter molecules or labels, which may be used forease of detection, include radionuclides, enzymes, fluorescent,chemiluminescent, or chromogenic agents as well as substrates,cofactors, inhibitors, magnetic particles, and the like.

[0127] Host cells transformed with nucleotide sequences encoding RCN maybe cultured under conditions suitable for the expression and recovery ofthe protein from cell culture. The protein produced by a transformedcell may be secreted or contained intracellularly depending on thesequence and/or the vector used. As will be understood by those of skillin the art, expression vectors containing polynucleotides which encodeRCN may be designed to contain signal sequences which direct secretionof RCN through a prokaryotic or eukaryotic cell membrane. Otherconstructions may be used to join sequences encoding RCN to nucleotidesequence encoding a polypeptide domain which will facilitatepurification of soluble proteins. Such purification facilitating domainsinclude, but are not limited to, metal chelating peptides such ashistidine-tryptophan modules that allow purification on immobilizedmetals, protein A domains that allow purification on immobilizedimmunoglobulin, and the domain utilized in the FLAGS extension/affinitypurification system (Immunex Corp., Seattle, Wash.). The inclusion ofcleavable linker sequences such as those specific for Factor XA orenterokinase (Invitrogen, San Diego, Calif.) between the purificationdomain and RCN may be used to facilitate purification. One suchexpression vector provides for expression of a fusion protein containingRCN and a nucleic acid encoding 6 histidine residues preceding athioredoxin or an enterokinase cleavage site. The histidine residuesfacilitate purification on IMIAC (immobilized metal ion affinitychromatography) as described in Porath, J. et al. (1992, Prot. Exp.Purif. 3: 263-281), while the enterokinase cleavage site provides ameans for purifying RCN from the fusion protein. A discussion of vectorswhich contain fusion proteins is provided in Kroll, D. J. et al. (1993;DNA Cell Biol. 12:441-453).

[0128] In addition to recombinant production, fragments of RCN may beproduced by direct peptide synthesis using solid-phase techniques(Merrifield J. (1963) J. Am. Chem. Soc. 85:2149-2154). Protein synthesismay be performed using manual techniques or by automation. Automatedsynthesis may be achieved, for example, using an Applied Biosystems 431Apeptide synthesizer (Perkin Elmer). Various fragments of RCN may bechemically synthesized separately and combined using chemical methods toproduce the full length molecule.

[0129] Therapeutics

[0130] Chemical and structural homology exists among RCN γ,reticulocalbin from human (GI 1262329), and clone REM#1 from rat (GI780361). In addition, RCN γ is expressed in lung, brain, breast, smoothmuscle, and endothelial cells; and at sites of hematopoiesis. Therefore,RCN γ appears to play a role in infectious, developmental, neoplastic,and immunological disorders where RCN γ is overexpressed.

[0131] Chemical and structural homology exists between RCN δ andreticulocalbin from human (GI 1262329). In addition, RCN δ is expressedin heart, gut, prostate, and smooth muscle; and at sites ofhematopoiesis. Therefore, RCN δ appears to play a role in infectious,developmental, neoplastic, and immunological disorders where RCN δ isoverexpressed.

[0132] Therefore, in one embodiment, RCN or a fragment or derivativethereof may be administered to a subject to treat an infectiousdisorder. An infectious disorder may include, but is not limited to,viral [adenoviruses (ARD, pneumonia), arenaviruses (lymphocyticchoriomeningitis), bunyaviruses (Hantavirus), coronaviruses (pneumonia,chronic bronchitis), hepadnaviruses (hepatitis), herpesviruses (HSV,VZV, Epstein-Barr virus, cytomegalovirus), flaviviruses ( yellow fever),orthomyxoviruses (influenza), papillomaviruses (cancer), paramyxoviruses(measles, mumps), picomoviruses (rhinovirus, poliovirus,coxsackie-virus), polyomaviruses (BK virus, JC virus), poxviruses(smallpox), reovirus (Colorado tick fever), retroviruses (HIV, HTLV),rhabdoviruses (rabies), rotaviruses (gastroenteritis), and togaviruses(encephalitis, rubella)], bacterial, fungal, parasitic, protozoal, orhelminthic infections.

[0133] In another embodiment, a vector capable of expressing RCN, or afragment or a derivative thereof, may also be administered to a subjectto treat an infectious disorder including, but not limited to, thosedescribed above.

[0134] In still another embodiment, an agonist of RCN may also beadministered to a subject to treat an infectious disorder including, butnot limited to, those described above.

[0135] In one embodiment, RCN or a fragment or derivative thereof may beadministered to a subject to treat a developmental disorder. The term“developmental disorder” refers to any disorder associated withdevelopment or function of a tissue, organ, or system of a subject,i.e., brain, adrenal gland, kidney, skeletal or reproductive system.Such disorders include, but are not limited to, renal tubular acidosis,anemia, Cushing's syndrome, achondroplastic dwarfism, epilepsy, gonadaldysgenesis, WAGR syndrome, hereditary neuropathies such asCharcot-Marie-Tooth disease and neurofibromatosis, hypothyroidism,hydrocephalus, seizure disorders such as Syndenham's chorea and cerebralpalsy, spinal bifida, and congenital glaucoma, cataract, orsensorineural hearing loss.

[0136] In another embodiment, a vector capable of expressing RCN, or afragment or a derivative thereof, may also be administered to a subjectto treat a developmental disorder including, but not limited to, thosedescribed above.

[0137] In still another embodiment, an agonist of RCN may also beadministered to a subject to treat a developmental disorder including,but not limited to, those described above.

[0138] In one embodiment, an antagonist of RCN may be administered to asubject to prevent or treat a neoplastic disorder. Such disorders mayinclude, but are not limited to, adenocarcinoma, leukemia, lymphoma,melanoma, myeloma, sarcoma, teratocarcinoma, and, particularly, cancersof the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix,gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver,lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivaryglands, skin, spleen, testis, thymus, thyroid, and uterus. In oneaspect, an antibody which specifically binds RCN may be used directly asan antagonist or indirectly as a targeting or delivery mechanism forbringing a pharmaceutical agent to cells or tissue which express RCN.

[0139] In another embodiment, a vector expressing the complement of thepolynucleotide encoding RCN may be administered to a subject to treat orprevent a neoplastic disorder including, but not limited to, thosedescribed above.

[0140] In another embodiment, an antagonist of RCN may be administeredto a subject to prevent or treat an immunological disorder. Suchdisorders may include, but are not limited to, AIDS, Addison's disease,adult respiratory distress syndrome, allergies, anemia, asthma,atherosclerosis, bronchitis, cholecystitis, Crohn's disease, ulcerativecolitis, atopic dermatitis, dermatomyositis, diabetes mellitus,emphysema, erythema nodosum, atrophic gastritis, glomerulonephritis,gout, Graves' disease, hypereosinophilia, irritable bowel syndrome,lupus erythematosus, multiple sclerosis, myasthenia gravis, myocardialor pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis,polymyositis, rheumatoid arthritis, scleroderma, Sjogren's syndrome,Werner syndrome, and autoimmune thyroiditis; complications of cancer,hemodialysis, extracorporeal circulation; viral, bacterial, fungal,parasitic, protozoal, and helminthic infections and trauma.

[0141] In another embodiment, a vector expressing the complement of thepolynucleotide encoding RCN may be administered to a subject to treat orprevent an immunological disorder including, but not limited to, thosedescribed above.

[0142] In other embodiments, any of the proteins, antagonists,antibodies, agonists, complementary sequences or vectors of theinvention may be administered in combination with other appropriatetherapeutic agents. Selection of the appropriate agents for use incombination therapy may be made by one of ordinary skill in the art,according to conventional pharmaceutical principles. The combination oftherapeutic agents may act synergistically to effect the treatment orprevention of the various disorders described above. Using thisapproach, one may be able to achieve therapeutic efficacy with lowerdosages of each agent, thus reducing the potential for adverse sideeffects.

[0143] An antagonist of RCN may be produced using methods which aregenerally known in the art. In particular, purified RCN may be used toproduce antibodies or to screen libraries of pharmaceutical agents toidentify those which specifically bind RCN.

[0144] Antibodies to RCN may be generated using methods that are wellknown in the art. Such antibodies may include, but are not limited to,polyclonal, monoclonal, chimeric, single chain, Fab fragments, andfragments produced by a Fab expression library. Neutralizing antibodies,(i.e., those which inhibit dimer formation) are especially preferred fortherapeutic use.

[0145] For the production of antibodies, various hosts including goats,rabbits, rats, mice, humans, and others, may be immunized by injectionwith RCN or any fragment or oligopeptide thereof which has immunogenicproperties. Depending on the host species, various adjuvants may be usedto increase immunological response. Such adjuvants include, but are notlimited to, Freund's, mineral gels such as aluminum hydroxide, andsurface active substances such as lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, anddinitrophenol. Among adjuvants used in humans, BCG (bacilliCalmette-Guerin) and Corynebacterium parvum are especially preferable.

[0146] It is preferred that the oligopeptides, peptides, or fragmentsused to induce antibodies to RCN have an amino acid sequence consistingof at least five amino acids and more preferably at least 10 aminoacids. It is also preferable that they are identical to a portion of theamino acid sequence of the natural protein, and they may contain theentire amino acid sequence of a small, naturally occurring molecule.Short stretches of RCN amino acids may be fused with those of anotherprotein such as keyhole limpet hemocyanin and antibody produced againstthe chimeric molecule.

[0147] Monoclonal antibodies to RCN may be prepared using any techniquewhich provides for the production of antibody molecules by continuouscell lines in culture. These include, but are not limited to, thehybridoma technique, the human B-cell hybridoma technique, and theEBV-hybridoma technique (Kohler, G. et al. (1975) Nature 256:495-497;Kozbor, D. et al. (1985) J. Immunol. Methods 81:31-42; Cote, R. J. etal. (1983) Proc. Natl. Acad. Sci. 80:2026-2030; Cole, S. P. et al.(1984) Mol. Cell Biol. 62:109-120).

[0148] In addition, techniques developed for the production of “chimericantibodies”, the splicing of mouse antibody genes to human antibodygenes to obtain a molecule with appropriate antigen specificity andbiological activity can be used (Morrison, S. L. et al. (1984) Proc.Natl. Acad. Sci. 81:6851-6855; Neuberger, M. S. et al. (1984) Nature312:604-608; Takeda, S. et al. (1985) Nature 314:452-454).Alternatively, techniques described for the production of single chainantibodies may be adapted, using methods known in the art, to produceRCN-specific single chain antibodies. Antibodies with relatedspecificity, but of distinct idiotypic composition, may be generated bychain shuffling from random combinatorial immunoglobin libraries (BurtonD. R. (1991) Proc. Natl. Acad. Sci. 88:11120-3).

[0149] Antibodies may also be produced by inducing in vivo production inthe lymphocyte population or by screening immunoglobulin libraries orpanels of highly specific binding reagents as disclosed in theliterature (Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci. 86:3833-3837; Winter, G. et al. (1991) Nature 349:293-299).

[0150] Antibody fragments which contain specific binding sites for RCNmay also be generated. For example, such fragments include, but are notlimited to, the F(ab′)2 fragments which can be produced by pepsindigestion of the antibody molecule and the Fab fragments which can begenerated by reducing the disulfide bridges of the F(ab′)2 fragments.Alternatively, Fab expression libraries may be constructed to allowrapid and easy identification of monoclonal Fab fragments with thedesired specificity (Huse, W. D. et al. (1989) Science 254:1275-1281).

[0151] Various immunoassays may be used for screening to identifyantibodies having the desired specificity. Numerous protocols forcompetitive binding or immunoradiometric assays using either polyclonalor monoclonal antibodies with established specificities are well knownin the art. Such immunoassays typically involve the measurement ofcomplex formation between RCN and its specific antibody. A two-site,monoclonal-based immunoassay utilizing monoclonal antibodies reactive totwo non-interfering RCN epitopes is preferred, but a competitive bindingassay may also be employed (Maddox, supra).

[0152] In another embodiment of the invention, the polynucleotidesencoding RCN, or any fragment or complement thereof, may be used fortherapeutic purposes. In one aspect, the complement of thepolynucleotide encoding RCN may be used in situations in which it wouldbe desirable to block the transcription of the mRNA. In particular,cells may be transformed with sequences complementary to polynucleotidesencoding RCN. Thus, complementary molecules or fragments may be used tomodulate RCN activity, or to achieve regulation of gene function. Suchtechnology is now well known in the art, and sense or antisenseoligonucleotides or larger fragments, can be designed from variouslocations along the coding or control regions of sequences encoding RCN.

[0153] Expression vectors derived from retro viruses, adenovirus, herpesor vaccinia viruses, or from various bacterial plasmids may be used fordelivery of nucleotide sequences to the targeted organ, tissue or cellpopulation. Methods which are well known to those skilled in the art canbe used to construct vectors which will express nucleic acid sequencewhich is complementary to the polynucleotides of the gene encoding RCN.These techniques are described both in Sambrook et al. (supra) and inAusubel et al. (supra).

[0154] Genes encoding RCN can be turned off by transforming a cell ortissue with expression vectors which express high levels of apolynucleotide or fragment thereof which encodes RCN. Such constructsmay be used to introduce untranslatable sense or antisense sequencesinto a cell. Even in the absence of integration into the DNA, suchvectors may continue to transcribe RNA molecules until they are disabledby endogenous nucleases. Transient expression may last for a month ormore with a non-replicating vector and even longer if appropriatereplication elements are part of the vector system.

[0155] As mentioned above, modifications of gene expression can beobtained by designing complementary sequences or antisense molecules(DNA, RNA, or PNA) to the control, 5′ or regulatory regions of the geneencoding RCN (signal sequence, promoters, enhancers, and introns).Oligonucleotides derived from the transcription initiation site, e.g.,between positions −10 and +10 from the start site, are preferred.Similarly, inhibition can be achieved using “triple helix” base-pairingmethodology. Triple helix pairing is useful because it causes inhibitionof the ability of the double helix to open sufficiently for the bindingof polymerases, transcription factors, or regulatory molecules. Recenttherapeutic advances using triplex DNA have been described in theliterature (Gee, J. E. et al. (1994) In: Huber, B. E. and B. I. Carr,Molecular and Immunologic Approaches, Futura Publishing Co., Mt. Kisco,N.Y.). The complementary sequence or antisense molecule may also bedesigned to block translation of mRNA by preventing the transcript frombinding to ribosomes.

[0156] Ribozymes, enzymatic RNA molecules, may also be used to catalyzethe specific cleavage of RNA. The mechanism of ribozyme action involvessequence-specific hybridization of the ribozyme molecule tocomplementary target RNA, followed by endonucleolytic cleavage. Exampleswhich may be used include engineered hammerhead motif ribozyme moleculesthat can specifically and efficiently catalyze endonucleolytic cleavageof sequences encoding RCN.

[0157] Specific ribozyme cleavage sites within any potential RNA targetare initially identified by scanning the target molecule for ribozymecleavage sites which include the following sequences: GUA, GUU, and GUC.Once identified, short RNA sequences of between 15 and 20ribonucleotides corresponding to the region of the target genecontaining the cleavage site may be evaluated for secondary structuralfeatures which may render the oligonucleotide inoperable. Thesuitability of candidate targets may also be evaluated by testingaccessibility to hybridization with complementary oligonucleotides usingribonuclease protection assays.

[0158] Complementary ribonucleic acid molecules and ribozymes of theinvention may be prepared by any method known in the art for thesynthesis of nucleic acid molecules. These include techniques forchemically synthesizing oligonucleotides such as solid phasephosphoramidite chemical synthesis. Alternatively, RNA molecules may begenerated by in vitro and in vivo transcription of DNA sequencesencoding RCN. Such DNA sequences may be incorporated into a wide varietyof vectors with suitable RNA polymerase promoters such as T7 or SP6.Alternatively, these cDNA constructs that synthesize complementary RNAconstitutively or inducibly can be introduced into cell lines, cells, ortissues.

[0159] RNA molecules may be modified to increase intracellular stabilityand half-life. Possible modifications include, but are not limited to,the addition of flanking sequences at the 5′ and/or 3′ ends of themolecule or the use of phosphorothioate or 2′O-methyl rather thanphosphodiesterase linkages within the backbone of the molecule. Thisconcept is inherent in the production of PNAs and can be extended in allof these molecules by the inclusion of nontraditional bases such asinosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-,and similarly modified forms of adenine, cytidine, guanine, thymine, anduridine which are not as easily recognized by endogenous endonucleases.

[0160] Many methods for introducing vectors into cells or tissues areavailable and equally suitable for use in vivo, in vitro, and ex vivo.For ex vivo therapy, vectors may be introduced into stem cells takenfrom the patient and clonally propagated for autologous transplant backinto that same patient. Delivery by transfection, by liposome injectionsor polycationic amino polymers (Goldman, C. K. et al. (1997) NatureBiotechnology 15:462-66; incorporated herein by reference) may beachieved using methods which are well known in the art.

[0161] Any of the therapeutic methods described above may be applied toany subject in need of such therapy, including, for example, mammalssuch as dogs, cats, cows, horses, rabbits, monkeys, and most preferably,humans.

[0162] An additional embodiment of the invention relates to theadministration of a pharmaceutical composition, in conjunction with apharmaceutically acceptable carrier, for any of the therapeutic effectsdiscussed above. Such pharmaceutical compositions may consist of RCN,antibodies to RCN, mimetics, agonists, antagonists, or inhibitors ofRCN. The compositions may be administered alone or in combination withat least one other agent, such as stabilizing compound, which may beadministered in any sterile, biocompatible pharmaceutical carrier,including, but not limited to, saline, buffered saline, dextrose, andwater. The compositions may be administered to a patient alone, or incombination with other agents, drugs or hormones.

[0163] The pharmaceutical compositions utilized in this invention may beadministered by any number of routes including, but not limited to,oral, intravenous, intramuscular, intra-arterial, intramedullary,intrathecal, intraventricular, transdermal, subcutaneous,intraperitoneal, intranasal, enteral, topical, sublingual, or rectalmeans.

[0164] In addition to the active ingredients, these pharmaceuticalcompositions may contain suitable pharmaceutically-acceptable carrierscomprising excipients and auxiliaries which facilitate processing of theactive compounds into preparations which can be used pharmaceutically.Further details on techniques for formulation and administration may befound in the latest edition of Remington's Pharmaceutical Sciences(Maack Publishing Co., Easton, Pa.).

[0165] Pharmaceutical compositions for oral administration can beformulated using pharmaceutically acceptable carriers well known in theart in dosages suitable for oral administration. Such carriers enablethe pharmaceutical compositions to be formulated as tablets, pills,dragees, capsules, liquids, gels, syrups, slurries, suspensions, and thelike, for ingestion by the patient.

[0166] Pharmaceutical preparations for oral use can be obtained throughcombination of active compounds with solid excipient, optionallygrinding a resulting mixture, and processing the mixture of granules,after adding suitable auxiliaries, if desired, to obtain tablets ordragee cores. Suitable excipients are carbohydrate or protein fillers,such as sugars, including lactose, sucrose, mannitol, or sorbitol;starch from corn, wheat, rice, potato, or other plants; cellulose, suchas methyl cellulose, hydroxypropylmethyl-cellulose, or sodiumcarboxymethylcellulose; gums including arabic and tragacanth; andproteins such as gelatin and collagen. If desired, disintegrating orsolubilizing agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, alginic acid, or a salt thereof, such as sodiumalginate.

[0167] Dragee cores may be used in conjunction with suitable coatings,such as concentrated sugar solutions, which may also contain gum arabic,talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/ortitanium dioxide, lacquer solutions, and suitable organic solvents orsolvent mixtures. Dyestuffs or pigments may be added to the tablets ordragee coatings for product identification or to characterize thequantity of active compound, i.e., dosage.

[0168] Pharmaceutical preparations which can be used orally includepush-fit capsules made of gelatin, as well as soft, sealed capsules madeof gelatin and a coating, such as glycerol or sorbitol. Push-fitcapsules can contain active ingredients mixed with a filler or binders,such as lactose or starches, lubricants, such as talc or magnesiumstearate, and, optionally, stabilizers. In soft capsules, the activecompounds may be dissolved or suspended in suitable liquids, such asfatty oils, liquid, or liquid polyethylene glycol with or withoutstabilizers.

[0169] Pharmaceutical formulations suitable for parenteraladministration may be formulated in aqueous solutions, preferably inphysiologically compatible buffers such as Hanks' solution, Ringer'ssolution, or physiologically buffered saline. Aqueous injectionsuspensions may contain substances which increase the viscosity of thesuspension, such as sodium carboxymethyl cellulose, sorbitol, ordextran. Additionally, suspensions of the active compounds may beprepared as appropriate oily injection suspensions. Suitable lipophilicsolvents or vehicles include fatty oils such as sesame oil, or syntheticfatty acid esters, such as ethyl oleate or triglycerides, or liposomes.Non-lipid polycationic amino polymers may also be used for delivery.Optionally, the suspension may also contain suitable stabilizers oragents which increase the solubility of the compounds to allow for thepreparation of highly concentrated solutions.

[0170] For topical or nasal administration, penetrants appropriate tothe particular barrier to be permeated are used in the formulation. Suchpenetrants are generally known in the art.

[0171] The pharmaceutical compositions of the present invention may bemanufactured in a manner that is known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping, or lyophilizing processes.

[0172] The pharmaceutical composition may be provided as a salt and canbe formed with many acids, including but not limited to, hydrochloric,sulfuric, acetic, lactic, tartaric, malic, succinic acids, etc. Saltstend to be more soluble in aqueous or other protonic solvents than arethe corresponding free base forms. In other cases, the preferredpreparation may be a lyophilized powder which may contain any or all ofthe following: 1-50 mM histidine, 0.1%-2% sucrose, and 2-7% mannitol, ata pH range of 4.5 to 5.5, that is combined with buffer prior to use.

[0173] After pharmaceutical compositions have been prepared, they can beplaced in an appropriate container and labeled for treatment of anindicated condition. For administration of RCN, such labeling wouldinclude amount, frequency, and method of administration.

[0174] Pharmaceutical compositions suitable for use in the inventioninclude compositions wherein the active ingredients are contained in aneffective amount to achieve the intended purpose. The determination ofan effective dose is well within the capability of those skilled in theart.

[0175] For any compound, the therapeutically effective dose can beestimated initially either in cell culture assays, e.g., of neoplasticcells, or in animal models, usually mice, rabbits, dogs, or pigs. Theanimal model may also be used to determine the appropriate concentrationrange and route of administration. Such information can then be used todetermine useful doses and routes for administration in humans.

[0176] A therapeutically effective dose refers to that amount of activeingredient, for example RCN or fragments thereof, antibodies of RCN,agonists, antagonists or inhibitors of RCN, which ameliorates thesymptoms or condition. Therapeutic efficacy and toxicity may bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., ED50 (the dose therapeutically effective in50% of the population) and LD50 (the dose lethal to 50% of thepopulation). The dose ratio of toxic to therapeutic effects is thetherapeutic index, and it can be expressed as the ratio, LD50/ED50.Pharmaceutical compositions which exhibit large therapeutic indices arepreferred. The data obtained from cell culture assays and animal studiesis used in formulating a range of dosage for human use. The dosagecontained in such compositions is preferably within a range ofcirculating concentrations that include the ED50 with little or notoxicity. The dosage varies within this range depending upon the dosageform employed, sensitivity of the patient, and the route ofadministration.

[0177] The exact dosage will be determined by the practitioner, in lightof factors related to the subject that requires treatment. Dosage andadministration are adjusted to provide sufficient levels of the activemoiety or to maintain the desired effect. Factors which may be takeninto account include the severity of the disease state, general healthof the subject, age, weight, and gender of the subject, diet, time andfrequency of administration, drug combination(s), reactionsensitivities, and tolerance/response to therapy. Long-actingpharmaceutical compositions may be administered every 3 to 4 days, everyweek, or once every two weeks depending on half-life and clearance rateof the particular formulation.

[0178] Normal dosage amounts may vary from 0.1 to 100,000 micrograms, upto a total dose of about 1 g, depending upon the route ofadministration. Guidance as to particular dosages and methods ofdelivery is provided in the literature and generally available topractitioners in the art. Those skilled in the art will employ differentformulations for nucleotides than for proteins or their inhibitors.Similarly, delivery of polynucleotides or polypeptides will be specificto particular cells, conditions, locations, etc.

[0179] Diagnostics

[0180] In another embodiment, antibodies which specifically bind RCN maybe used for the diagnosis of conditions or diseases characterized byexpression of RCN, or in assays to monitor patients being treated withRCN, agonists, antagonists or inhibitors. The antibodies useful fordiagnostic purposes may be prepared in the same manner as thosedescribed above for therapeutics. Diagnostic assays for RCN includemethods which utilize the antibody and a label to detect RCN in humanbody fluids or extracts of cells or tissues. The antibodies may be usedwith or without modification, and may be labeled by joining them, eithercovalently or non-covalently, with a reporter molecule. A wide varietyof reporter molecules which are known in the art may be used, several ofwhich are described above.

[0181] A variety of protocols for measuring RCN, including ELSA, RIA,and FACS, are known in the art and provide a basis for diagnosingaltered or abnormal levels of RCN expression. Normal or standard valuesfor RCN expression are established by combining body fluids or cellextracts taken from normal mammalian subjects, preferably human, withantibody to RCN under conditions suitable for complex formation. Theamount of standard complex formation may be quantified by variousmethods, preferably by photometric, means. Quantities of RCN expressedin subject samples, samples from biopsied tissues are compared with thestandard values. Deviation between standard and subject valuesestablishes the parameters for diagnosing disease.

[0182] In another embodiment of the invention, the polynucleotidesencoding RCN may be used for diagnostic purposes. The polynucleotideswhich may be used include oligonucleotide sequences, complementary RNAand DNA molecules, and PNAs. The polynucleotides may be used to detectand quantitate gene expression in biopsied tissues in which expressionof RCN may be correlated with disease. The diagnostic assay may be usedto distinguish between absence, presence, and excess expression of RCN,and to monitor regulation of RCN levels during therapeutic intervention.

[0183] In one aspect, hybridization with PCR probes which are capable ofdetecting polynucleotide sequences, including genomic sequences,encoding RCN or closely related molecules, may be used to identifynucleic acid sequences which encode RCN. The specificity of the probe,whether it is made from a highly specific region, e.g., 10 uniquenucleotides in the 5′ regulatory region, or a less specific region,e.g., especially in the 3′ coding region, and the stringency of thehybridization or amplification (maximal, high, intermediate, or low)will determine whether the probe identifies only naturally occurringsequences encoding RCN, alleles, or related sequences.

[0184] Probes may also be used for the detection of related sequences,and should preferably contain at least 50% of the nucleotides from anyof the RCN encoding sequences. The hybridization probes of the subjectinvention may be DNA or RNA and derived from the nucleotide sequence ofSEQ ID NO:2 or SEQ ID NO:4 or from genomic sequence including promoter,enhancer elements, and introns of the naturally occurring RCN.

[0185] Means for producing specific hybridization probes for DNAsencoding RCN include the cloning of nucleic acid sequences encoding RCNor RCN derivatives into vectors for the production of mRNA probes. Suchvectors are known in the art and are, commercially available, and may beused to synthesize RNA probes in vitro by means of the addition of theappropriate RNA polymerases and the appropriate labeled nucleotides.Hybridization probes may be labeled by a variety of reporter groups, forexample, radionuclides such as 32P or 35S, or enzymatic labels, such asalkaline phosphatase coupled to the probe via avidin/biotin couplingsystems, and the like.

[0186] Polynucleotide sequences encoding RCN may be used for thediagnosis of conditions or disorders which are associated withexpression of RCN. Examples of such conditions or disorders include aninfectious disorder, such as viral [adenoviruses (ARD, pneumonia),arenaviruses (lymphocytic choriomeningitis), bunyaviruses (Hantavirus),coronaviruses (pneumonia, chronic bronchitis), hepadnaviruses(hepatitis), herpesviruses (HSV, VZV, Epstein-Barr virus,cytomegalovirus), flaviviruses ( yellow fever), orthomyxoviruses(influenza), papillomaviruses (cancer), paramyxoviruses (measles,mumps), picomoviruses (rhinovirus, poliovirus, coxsackie-virus),polyomaviruses (BK virus, JC virus), poxviruses (smallpox), reovirus(Colorado tick fever), retroviruses (HIV, HTLV), rhabdoviruses (rabies),rotaviruses (gastroenteritis), and togaviruses (encephalitis, rubella)],bacterial, fungal, parasitic, protozoal, or helminthic infections; adevelopmental disorder, such as renal tubular acidosis, anemia,Cushing's syndrome, achondroplastic dwarfism, epilepsy, gonadaldysgenesis, WAGR syndrome, hereditary neuropathies such asCharcot-Marie-Tooth disease and neurofibromatosis, hypothyroidism,hydrocephalus, seizure disorders (such as Syndenham's chorea andcerebral palsy), spinal bifida, congenital glaucoma, cataract, orsensorineural hearing loss; a neoplastic disorder, such asadenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma,teratocarcinoma, and, particularly, cancers of the adrenal gland,bladder, bone, bone marrow, brain, breast, cervix, gall bladder,ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle,ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin,spleen, testis, thymus, thyroid, and uterus; and an immunologicaldisorder, such as AIDS, Addison's disease, adult respiratory distresssyndrome, allergies, anemia, asthma, atherosclerosis, bronchitis,cholecystitis, Crohn's disease, ulcerative colitis, atopic dermatitis,dermatomyositis, diabetes mellitus, emphysema, erythema nodosum,atrophic gastritis, glomerulonephritis, gout, Graves' disease,hypereosinophilia, irritable bowel syndrome, lupus erythematosus,multiple sclerosis, myasthenia gravis, myocardial or pericardialinflammation, osteoarthritis, osteoporosis, pancreatitis, polymyositis,rheumatoid arthritis, scleroderma, Sjogren's syndrome, Werner syndrome,and autoimmune thyroiditis; complications of cancer, hemodialysis, andextracorporeal circulation. The polynucleotide sequences encoding RCNmay be used in Southern or northern analysis, dot blot, or othermembrane-based technologies; in PCR technologies; or in dipstick, pin,ELISA assays or microarrays utilizing fluids or tissues from patientbiopsies to detect altered RCN expression. Such qualitative orquantitative methods are well known in the art.

[0187] In a particular aspect, the nucleotide sequences encoding RCN maybe useful in assays that detect activation or induction of variouscancers, particularly those mentioned above. The nucleotide sequencesencoding RCN may be labeled by standard methods, and added to a fluid ortissue sample from a patient under conditions suitable for the formationof hybridization complexes. After a suitable incubation period, thesample is washed and the signal is quantitated and compared with astandard value. If the amount of signal in the biopsied or extractedsample is significantly altered from that of a comparable controlsample, the nucleotide sequences have hybridized with nucleotidesequences in the sample, and the presence of altered levels ofnucleotide sequences encoding RCN in the sample indicates the presenceof the associated disease. Such assays may also be used to evaluate theefficacy of a particular therapeutic treatment regimen in animalstudies, in clinical trials, or in monitoring the treatment of anindividual patient.

[0188] In order to provide a basis for the diagnosis of diseaseassociated with expression of RCN, a normal or standard profile forexpression is established. This may be accomplished by combining bodyfluids or cell extracts taken from normal subjects, either animal orhuman, with a sequence, or a fragment thereof, which encodes RCN, underconditions suitable for hybridization or amplification. Standardhybridization may be quantified by comparing the values obtained fromnormal subjects with those from an experiment where a known amount of asubstantially purified polynucleotide is used. Standard values obtainedfrom normal samples may be compared with values obtained from samplesfrom patients who are symptomatic for disease. Deviation betweenstandard and subject values is used to establish the presence ofdisease.

[0189] Once disease is established and a treatment protocol isinitiated, hybridization assays may be repeated on a regular basis toevaluate whether the level of expression in the patient begins toapproximate that which is observed in the normal patient. The resultsobtained from successive assays may be used to show the efficacy oftreatment over a period ranging from several days to months.

[0190] With respect to cancer, the presence of a relatively high amountof transcript in biopsied tissue from an individual may indicate apredisposition for the development of the disease, or may provide ameans for detecting the disease prior to the appearance of actualclinical symptoms. A more definitive diagnosis of this type may allowhealth professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the cancer.

[0191] Additional diagnostic uses for oligonucledtides designed from thesequences encoding RCN may involve the use of PCR. Such oligomers may bechemically synthesized, generated enzymatically, or produced in vitro.Oligomers will preferably consist of two nucleotide sequences, one withsense orientation (5′−>3′) and another with antisense (3′<−5′), employedunder optimized conditions for identification of a specific gene orcondition. The same two oligomers, nested sets of oligomers, or even adegenerate pool of oligomers may be employed under less stringentconditions for detection and/or quantitation of closely related DNA orRNA sequences.

[0192] Methods which may also be used to quantitate the expression ofRCN include radiolabeling or biotinylating nucleotides, coamplificationof a control nucleic acid, and standard curves onto which theexperimental results are interpolated (Melby, P. C. et al. (1993) J.Immunol. Methods, 159:235-244; Duplaa, C. et al. (1993) Anal. Biochem.212:229-236). The speed of quantitation of multiple samples may beaccelerated by running the assay in an ELISA format where the oligomerof interest is presented in various dilutions and a spectrophotometricor calorimetric response gives rapid quantitation.

[0193] In further embodiments, an oligonucleotide derived from any ofthe polynucleotide sequences described herein may be used as a target ina microarray. The microarray can be used to monitor the expression levelof large numbers of genes simultaneously (to produce a transcriptimage), and to identify genetic variants, mutations and polymorphisms.This information will be useful in determining gene function,understanding the genetic basis of disease, diagnosing disease, and indeveloping and monitoring the activity of therapeutic agents (Heller, R.et al. (1997) Proc. Natl. Acad. Sci. 94:2150-55) .

[0194] In one embodiment, the microarray is prepared and used accordingto the methods described in PCT application W095/11995 (Chee et al.),Lockhart, D. J. et al. (1996; Nat. Biotech. 14: 1675-1680) and Schena,M. et al. (1996; Proc. Natl. Acad. Sci. 93: 10614-10619), all of whichare incorporated herein in their entirety by reference.

[0195] The microarray is preferably composed of a large number ofunique, single-stranded nucleic acid sequences, usually either syntheticantisense oligonucleotides or fragments of cDNAs, fixed to a solidsupport. The oligonucleotides are preferably about 6-60 nucleotides inlength, more preferably 15-30 nucleotides in length, and most preferablyabout 20-25 nucleotides in length. For a certain type of microarray, itmay be preferable to use oligonucleotides which are only 7-10nucleotides in length. The microarray may contain oligonucleotides whichcover the known 5′, or 3′, sequence, sequential oligonucleotides whichcover the full length sequence; or unique oligonucleotides selected fromparticular areas along the length of the sequence. Polynucleotides usedin the microarray may be oligonucleotides that are specific to a gene orgenes of interest in which at least a fragment of the sequence is knownor that are specific to one or more unidentified cDNAs which are commonto a particular cell type, developmental or disease state.

[0196] In order to produce oligonucleotides to a known sequence for amicroarray, the gene of interest is examined using a computer algorithmwhich starts at the 5′ or more preferably at the 3′ end of thenucleotide sequence. The algorithm identifies oligomers of definedlength that are unique to the gene, have a GC content within a rangesuitable for hybridization, and lack predicted secondary structure thatmay interfere with hybridization. In certain situations it may beappropriate to use pairs of oligonucleotides on a microarray. The“pairs” will be identical, except for one nucleotide which preferably islocated in the center of the sequence. The second oligonucleotide in thepair (mismatched by one) serves as a control. The number ofoligonucleotide pairs may range from two to one million. The oligomersare synthesized at designated areas on a substrate using alight-directed chemical process. The substrate may be paper, nylon orother type of membrane, filter, chip, glass slide or any other suitablesolid support.

[0197] In another aspect, an oligonucleotide may be synthesized on thesurface of the substrate by using a chemical coupling procedure and anink jet application apparatus, as described in PCT applicationWO95/251116 (Baldeschweiler et al.) which is incorporated herein in itsentirety by reference. In another aspect, a “gridded” array analogous toa dot (or slot) blot may be used to arrange and link cDNA fragments oroligonucleotides to the surface of a substrate using a vacuum system,thermal, UV, mechanical or chemical bonding procedures. An array, suchas those described above, may be produced by hand or by using availabledevices (slot blot or dot blot apparatus), materials (any suitable solidsupport), and machines (including robotic instruments), and may contain8, 24, 96, 384, 1536 or 6144 oligonucleotides, or any other numberbetween two and one million which lends itself to the efficient use ofcommercially available instrumentation.

[0198] In order to conduct sample analysis using a microarray, the RNAor DNA from a biological sample is made into hybridization probes. ThemRNA is isolated, and cDNA is produced and used as a template to makeantisense RNA (aRNA). The aRNA is amplified in the presence offluorescent nucleotides, and labeled probes are incubated with themicroarray so that the probe sequences hybridize to complementaryoligonucleotides of the microarray. Incubation conditions are adjustedso that hybridization occurs with precise complementary matches or withvarious degrees of less complementarity. After removal of nonhybridizedprobes, a scanner is used to determine the levels and patterns offluorescence. The scanned images are examined to determine degree ofcomplementarity and the relative abundance of each oligonucleotidesequence on the microarray. The biological samples may be obtained fromany bodily fluids (such as blood, urine, saliva, phlegm, gastric juices,etc.), cultured cells, biopsies, or other tissue preparations. Adetection system may be used to measure the absence, presence, andamount of hybridization for all of the distinct sequencessimultaneously. This data may be used for large scale correlationstudies on the sequences, mutations, variants, or polymorphisms amongsamples.

[0199] In another embodiment of the invention, the nucleic acidsequences which encode RCN may also be used to generate hybridizationprobes which are useful for mapping the naturally occurring genomicsequence. The sequences may be mapped to a particular chromosome, to aspecific region of a chromosome or to artificial chromosomeconstructions, such as human artificial chromosomes (HACs), yeastartificial chromosomes (YACs), bacterial artificial chromosomes (BACs),bacterial P1 constructions or single chromosome cDNA libraries asreviewed in Price, C. M. (1993) Blood Rev. 7:127-134, and Trask, B. J.(1991) Trends Genet. 7:149-154.

[0200] Fluorescent in situ hybridization (FISH as described in Verma etal. (1988) Human Chromosomes: A Manual of Basic Techniques, PergamonPress, New York, N.Y.) may be correlated with other physical chromosomemapping techniques and genetic map data. Examples of genetic map datacan be found in various scientific journals or at Online MendelianInheritance in Man (OMIM). Correlation between the location of the geneencoding RCN on a physical chromosomal map and a specific disease , orpredisposition to a specific disease, may help delimit the region of DNAassociated with that genetic disease. The nucleotide sequences of thesubject invention may be used to detect differences in gene sequencesbetween normal, carrier, or affected individuals.

[0201] In situ hybridization of chromosomal preparations and physicalmapping techniques such as linkage analysis using establishedchromosomal markers may be used for extending genetic maps. Often theplacement of a gene on the chromosome of another mammalian species, suchas mouse, may reveal associated markers even if the number or arm of aparticular human chromosome is not known. New sequences can be assignedto chromosomal arms, or parts thereof, by physical mapping. Thisprovides valuable information to investigators searching for diseasegenes using positional cloning or other gene discovery techniques. Oncethe disease or syndrome has been crudely localized by genetic linkage toa particular genomic region, for example, AT to 11q22-23 (Gatti, R. A.et al. (1988) Nature 336:577-580), any sequences mapping to that areamay represent associated or regulatory genes for further investigation.The nucleotide sequence of the subject invention may also be used todetect differences in the chromosomal location due to translocation,inversion, etc. among normal, carrier, or affected individuals.

[0202] In another embodiment of the invention, RCN, its catalytic orimmunogenic fragments or oligopeptides thereof, can be used forscreening libraries of compounds in any of a variety of drug screeningtechniques. The fragment employed in such screening may be free insolution, affixed to a solid support, borne on a cell surface, orlocated intracellularly. The formation of binding complexes, between RCNand the agent being tested, may be measured.

[0203] Another technique for drug screening which may be used providesfor high throughput screening of compounds having suitable bindingaffinity to the protein of interest as described in published PCTapplication WO84/03564. In this method, as applied to RCN large numbersof different small test compounds are synthesized on a solid substrate,such as plastic pins or some other surface. The test compounds arereacted with RCN, or fragments thereof, and washed. Bound RCN is thendetected by methods well known in the art. Purified RCN can also becoated directly onto plates for use in the aforementioned drug screeningtechniques. Alternatively, non-neutralizing antibodies can be used tocapture the peptide and immobilize it on a solid support.

[0204] In another embodiment, one may use competitive drug screeningassays in which neutralizing antibodies capable of binding RCNspecifically compete with a test compound for binding RCN. In thismanner, the antibodies can be used to detect the presence of any peptidewhich shares one or more antigenic determinants with RCN.

[0205] In additional embodiments, the nucleotide sequences which encodeRCN may be used in any molecular biology techniques that have yet to bedeveloped, provided the new techniques rely on properties of nucleotidesequences that are currently known, including, but not limited to, suchproperties as the triplet genetic code and specific base pairinteractions.

[0206] The examples below are provided to illustrate the subjectinvention and are not included for the purpose of limiting theinvention.

EXAMPLES

[0207] I cDNA Library Construction

[0208] RATRNOT02

[0209] The right atrium tissue used for the RATRNOT02 libraryconstruction was obtained from a 39 year old Caucasian male who died ofa gun shot wound. The patient had no history of heart disease,hypertension, cancer, diabetes or liver disease.

[0210] The frozen tissue was homogenized and lysed using a PolytronPT-3000 homogenizer (Brinkmann Instruments, Westbury N.J.) inguanidinium isothiocyanate solution. The lysate was centrifuged over a5.7 M CsCl cushion using an SW28 rotor in an L8-70M ultracentrifuge(Beckman Instruments) for 18 hours at 25,000 rpm at ambient temperature.The RNA was extracted with phenol chloroform pH 4.0, precipitated using0.3 M sodium acetate and 2.5 volumes of ethanol, resuspended inRNAse-free water and treated with DNase at 37° C. Extraction andprecipitation were repeated as before. The mRNA was then isolated withthe OLIGOTEX kit (QIAGEN Inc., Chatsworth Calif.) and used to constructthe cDNA library. A 10 million clone cDNA library was constructed usingthree micrograms of poly A⁺ mRNA and Not I/oligo d(T) primer. The cDNAswere directionally inserted into Sal I/Not I sites of PSPORT1(GIBco-BRL, Gaithersburg Md.).

[0211] BLADNOT03

[0212] The BLADNOT03 cDNA library was constructed from microscopicallynormal bladder tissue obtained from a 80-year-old Caucasian female. Thenormal tissue from the anterior wall was excised along with the tumoroustissue during a radical cysterectomy of a grade 3 of 4 invasivetransitional cell carcinoma located on the posterior wall. Prior tosurgery the patient had a history of a malignant neoplasm of the uterus,a total hysterectomy, removal of the fallopian tubes and ovaries,partial thyroidectomy, aorto-coronary bypass, hypertension, andatherosclerosis. There was a family history of atherosclerosis in thefather and a sibling, and osteoarthritis in the mother.

[0213] The frozen tissue was homogenized and lysed using a PolytronPT-3000 homogenizer (Brinkmann Instruments, Westbury, N.J.) inguanidinium isothiocyanate solution. The lysate was centrifuged over a5.7 M CsCl cushion using an SW28 rotor in a Beckman L8-70MUltracentrifuge (Beckman Instruments) for 18 hours at 25,000 rpm atambient temperature. The RNA was extracted with acid phenol pH 4.7,precipitated using 0.3 M sodium acetate and 2.5 volumes of ethanol,resuspended in RNAse-free water, and treated with DNase at 37° C.Extraction and precipitation were repeated as before. The mRNA was thenisolated with the OLIGOTEX kit (QIAGEN Inc.; Chatsworth, Calif.) andused to construct the cDNA library.

[0214] The mRNA was handled according to the recommended protocols inthe SUPERSCRIPT plasmid system (Cat. #18248-013; GIBCO-BRL). cDNAs werefractionated on a SEPHAROSE CL4B column (Cat. #275105-01; Pharmacia),and those cDNAs exceeding 400 bp were ligated to EcoRI adaptors,digested with NotI, size selected, and cloned into the NotI and EcoRIsites of pINCY vector (Incyte). The plasmid pINCY was subsequentlytransformed into DH5α competent cells (Cat. #18258-012; GIBCO-BRL).

[0215] II Isolation and Sequencing of cDNA Clones

[0216] RATRNOT02

[0217] Plasmid DNA was released from the cells and purified using theMINIPREP Kit (Catalog #77468; Advanced Genetic Technologies Corporation,Gaithersburg Md.). This kit consists of a 96-well block with reagentsfor 960 purifications. The recommended protocol was employed except forthe following changes: 1) the 96 wells were each filled with only 1 mlof sterile Terrific Broth (Catalog #22711, GIBCO-BRL) with carbenicillinat 25 mg/L and glycerol at 0.4%; 2) the bacteria were cultured for 24hours after the wells were inoculated and then lysed with 60 μl of lysisbuffer; 3) a centrifugation step employing the Beckman GS-6R rotor at2900 rpm for 5 minutes was performed before the contents of the blockwere added to the primary filter plate; and 4) the optional step ofadding isopropanol to TRIS buffer was not routinely performed. After thelast step in the protocol, samples were transferred to a Beckman 96-wellblock for storage.

[0218] The cDNAs were sequenced by the method of Sanger F and A RCoulson (1975; J Mol Biol 94:441f), using a Hamilton Micro Lab 2200(Hamilton, Reno Nev.) in combination with Peltier Thermal Cyclers(PTC200 from M J Research, Watertown Mass.) and Applied Biosystems 377DNA sequencing systems; and the reading frame was determined.

[0219] BLADNOT03

[0220] Plasmid DNA was released from the cells and purified using theR.E.A.L. PREP 96 plasmid kit (Catalog #26173; QIAGEN, Inc.). This kitenabled the simultaneous purification of 96 samples in a 96-well blockusing multi-channel reagent dispensers. The recommended protocol wasemployed except for the following changes: 1) the bacteria were culturedin 1 ml of sterile Terrific Broth (Catalog #22711, GIBCO-BRL) withcarbenicillin at 25 mg/L and glycerol at 0.4%; 2) after inoculation, thecultures were incubated for 19 hours and at the end of incubation, thecells were lysed with 0.3 ml of lysis buffer; and 3) followingisopropanol precipitation, the plasmid DNA pellet was resuspended in 0.1ml of distilled water. After the last step in the protocol, samples weretransferred to a 96-well block for storage at 4° C.

[0221] The cDNAs were sequenced by the method of Sanger et al. (1975, J.Mol. Biol. 94:441f), using a MICROLAB 2200 (Hamilton, Reno, Nev.) incombination with Peltier thermal cyclers (PTC200 from M J Research,Watertown, Mass.), and Applied Biosystems 377 DNA sequencing systems,and the reading frame was determined.

[0222] III Homology Searching of cDNA Clones and Their Deduced Proteins

[0223] The nucleotide sequences of the Sequence Listing or amino acidsequences deduced from them were used as query sequences againstdatabases such as GenBank, SwissProt, BLOCKS, and Pima II. Thesedatabases which contain previously identified and annotated sequenceswere searched for regions of homology (similarity) using BLAST, whichstands for Basic Local Alignment Search Tool (Altschul, S. F. (1993) J.Mol. Evol. 36:290-300; Altschul et al. (1990) J. Mol. Biol.215:403-410).

[0224] BLAST produces alignments of both nucleotide and amino acidsequences to determine sequence similarity. Because of the local natureof the alignments, BLAST is especially useful in determining exactmatches or in identifying homologs which may be of prokaryotic(bacterial) or eukaryotic (animal, fungal or plant) origin. Otheralgorithms such as the one described in Smith R. F. and T. F. Smith(1992; Protein Engineering 5:35-51), incorporated herein by reference,can be used when dealing with primary sequence patterns and secondarystructure gap penalties. As disclosed in this application, the sequenceshave lengths of at least 49 nucleotides, and no more than 12% uncalledbases (where N is recorded rather than A, C, G, or T).

[0225] The BLAST approach, as detailed in Karlin, S. and S. F. Atschul(1993; Proc. Nat. Acad. Sci. 90:5873-7) and incorporated herein byreference, involves searches for matches between a query sequence and adatabase sequence to evaluate the statistical significance of anymatches found and to report only those matches which satisfy theuser-selected threshold of significance. In this application, thresholdwas set at 10⁻²⁵ for nucleotides and 10⁻¹⁴ for peptides.

[0226] Incyte nucleotide sequences were searched against the GenBankdatabases for primate (pri), rodent (rod), and mammalian sequences(mam), and deduced amino acid sequences from the same clones weresearched against GenBank functional protein databases, mammalian (mamp),vertebrate (vrtp) and eukaryote (eukp), for homology.

[0227] IV Northern Analysis

[0228] Northern analysis is a laboratory technique used to detect thepresence of a transcript of a gene. Northern analysis involves thehybridization of a labeled nucleotide sequence to a membrane on whichRNAs from a particular cell type or tissue have been bound (Sambrook etal., supra).

[0229] Analogous computer techniques using BLAST (Altschul, S. F. (1993)J. Mol. Evol. 36:290-300; Altschul, S. F. et al. (1990) J. Mol. Evol.215:403-410) are used to search for identical or related molecules innucleotide databases such as GenBank or the LIFESEQ (IncytePharmaceuticals). This analysis is much faster than multiple,membrane-based hybridizations. In addition, the sensitivity of thecomputer search can be modified to determine whether any particularmatch is categorized as exact or homologous.

[0230] The basis of the search is the product score which is defined as:$\frac{\% \quad {sequence}\quad {identity} \times \% \quad {maximum}\quad {BLAST}\quad {score}}{100}$

[0231] The product score takes into account both the degree ofsimilarity between two sequences and the length of the sequence match.For example, with a product score of 40, the match will be exact withina 1-2% error; and at 70, the match will be exact. Homologous moleculesare usually identified by selecting those which show product scoresbetween 15 and 40, although lower scores may identify related molecules.

[0232] The results of northern analysis are reported as a list oflibraries in which the transcript encoding RCN occurs. Abundance andpercent abundance are also reported. Abundance directly reflects thenumber of times a particular transcript is represented in a cDNAlibrary, and percent abundance is abundance divided by the total numberof sequences examined in the cDNA library.

[0233] V Extension of RCN Encoding Polynucleotides

[0234] The nucleic acid sequences of Incyte Clones 922578 or 1601793were used to design oligonucleotide primers for extending a partialnucleotide sequence to full length. One primer was synthesized toinitiate extension in the antisense direction, and the other wassynthesized to extend sequence in the sense direction. Primers were usedto facilitate the extension of the known sequence “outward” generatingamplicons containing new, unknown nucleotide sequence for the region ofinterest. The initial primers were designed from the cDNA using OLIGO4.06 software (National Biosciences), or another appropriate program, tobe about 22 to about 30 nucleotides in length, to have a GC content of50% or more, and to anneal to the target sequence at temperatures ofabout 68° to about 72° C. Any stretch of nucleotides which would resultin hairpin structures and primer-primer dimerizations was avoided.

[0235] Selected human cDNA libraries (Gibco/BRL) were used to extend thesequence If more than one extension is necessary or desired, additionalsets of primers are designed to further extend the known region.

[0236] High fidelity amplification was obtained by following theinstructions for the XL-PCR kit (Perkin Elmer) and thoroughly mixing theenzyme and reaction mix. Beginning with 40 pmol of each primer and therecommended concentrations of all other components of the kit, PCR wasperformed using the Peltier thermal cycler (PTC200; M. J. Research,Watertown, Mass.) and the following parameters: Step 1 94° C. for 1 min(initial denaturation) Step 2 65° C. for 1 min Step 3 68° C. for 6 minStep 4 94° C. for 15 sec Step 5 65° C. for 1 min Step 6 68° C. for 7 minStep 7 Repeat step 4-6 for 15 additional cycles Step 8 94° C. for 15 secStep 9 65° C. for 1 min Step 10 68° C. for 7:15 min Step 11 Repeat step8-10 for 12 cycles Step 12 72° C. for 8 min Step 13 4° C. (and holding)

[0237] A 5-10 μl aliquot of the reaction mixture was analyzed byelectrophoresis on a low concentration (about 0.6-0.8%) agarose mini-gelto determine which reactions were successful in extending the sequence.Bands thought to contain the largest products were excised from the gel,purified using QIAQUICK (QIAGEN Inc., Chatsworth, Calif.), and trimmedof overhangs using Klenow enzyme to facilitate religation and cloning.

[0238] After ethanol precipitation, the products were redissolved in 13μl of ligation buffer, 1 μl T4-DNA ligase (15 units) and 1 μl T4polynucleotide kinase were added, and the mixture was incubated at roomtemperature for 2-3 hours or overnight at 16° C. Competent E. coli cells(in 40 μl of appropriate media) were transformed with 3 μl of ligationmixture and cultured in 80 μl of SOC medium (Sambrook et al., supra).After incubation for one hour at 37° C., the E. coli mixture was platedon Luria Bertani (LB)-agar (Sambrook et al., supra) containing 2×Carb.The following day, several colonies were randomly picked from each plateand cultured in 150 μl of liquid LB/2×Carb medium placed in anindividual well of an appropriate, commercially-available, sterile96-well microtiter plate. The following day, 5 μl of each overnightculture was transferred into a non-sterile 96-well plate and afterdilution 1:10 with water, 5 μl of each sample was transferred into a PCRarray.

[0239] For PCR amplification, 18 μl of concentrated PCR reaction mix(3.3×) containing 4 units of rTth DNA polymerase, a vector primer, andone or both of the gene specific primers used for the extension reactionwere added to each well. Amplification was performed using the followingconditions: Step 1 94° C. for 60 sec Step 2 94° C. for 20 sec Step 3 55°C. for 30 sec Step 4 72° C. for 90 sec Step 5 Repeat steps 2-4 for anadditional 29 cycles Step 6 72° C. for 180 sec Step 7 4° C. (andholding)

[0240] Aliquots of the PCR reactions were run on agarose gels togetherwith molecular weight markers. The sizes of the PCR products werecompared to the original partial cDNAs, and appropriate clones wereselected, ligated into plasmid, and sequenced.

[0241] In like manner, the nucleotide sequence of SEQ ID NO:2, or SEQ IDNO:4, is used to obtain 5′ regulatory sequences using the procedureabove, oligonucleotides designed for 5′ extension, and an appropriategenomic library.

[0242] VI Labeling and Use of Individual Hybridization Probes

[0243] Hybridization probes derived from SEQ ID NO:2 or SEQ ID NO:4 areemployed to screen cDNAs, genomic DNAs, or mRNAs. Although the labelingof oligonucleotides, consisting of about 20 base-pairs, is specificallydescribed, essentially the same procedure is used with larger nucleotidefragments. Oligonucleotides are designed using state-of-the-art softwaresuch as OLIGO 4.06 software (National Biosciences) and are labeled bycombining 50 pmol of each oligomer and 250 μCi of [γ-³²P] adenosinetriphosphate (Amersham) and T4 polynucleotide kinase (DuPont NEN,Boston, Mass.). The labeled oligonucleotides are substantially purifiedwith SEPHADEX G-25 superfine resin column (Pharmacia & Upjohn). Aaliquot containing 10⁷ counts per minute of the labeled probe is used ina typical membrane-based hybridization analysis of human genomic DNAdigested with one of the following endonucleases (Ase I, Bgl II, Eco RI,Pst I, Xba 1, or Pvu II; DuPont NEN).

[0244] The DNA from each digest is fractionated on a 0.7 percent agarosegel and transferred to nylon membranes (N.Y. TRAN Plus, Schleicher &Schuell, Durham, N.H.). Hybridization is carried out for 16 hours at 40°C. To remove nonspecific signals, blots are sequentially washed at roomtemperature under increasingly stringent conditions up to 0.1×salinesodium citrate and 0.5% sodium dodecyl sulfate. After XOMAT AR film(Kodak, Rochester, N.Y.) is exposed to the blots or the blots areexposed in a Phosphoimager cassette (Molecular Dynamics, Sunnyvale,Calif.) for several hours, hybridization patterns are compared visually.

[0245] VII Microarrays

[0246] To produce oligonucleotides for a microarray, one of thenucleotide sequences described herein is examined using a computeralgorithm which starts at the 3′ end of the nucleotide sequence. Thealgorithm identifies oligomers of defined length that are unique to thegene, have a GC content within a range suitable for hybridization, andlack predicted secondary structure that would interfere withhybridization. The algorithm identifies 20 sequence-specificoligonucleotides of 20 nucleotides in length (20-mers). A matched set ofoligonucleotides is created in which one nucleotide in the center ofeach sequence is altered. This process is repeated for each gene in themicroarray, and double sets of twenty 20 mers are synthesized andarranged on the surface of the silicon chip using a light-directedchemical process (Chee, M. et al., PCT/WO95/11995, incorporated hereinby reference).

[0247] In the alternative, a chemical coupling procedure and an ink jetdevice are used to synthesize oligomers on the surface of a substrate(Baldeschweiler, J. D. et al., PCTJWO95/25116, incorporated herein byreference). In another alternative, a “gridded” array analogous to a dot(or slot) blot is used to arrange and link cDNA fragments oroligonucleotides to the surface of a substrate using a vacuum system,thermal, UV, mechanical or chemical bonding procedures. An array may beproduced by hand or using available materials and machines and containgrids of 8 dots, 24 dots, 96 dots, 384 dots, 1536 dots or 6144 dots.After hybridization, the microarray is washed to remove nonhybridizedprobes, and a scanner is used to determine the levels and patterns offluorescence. The scanned images are examined to determine degree ofcomplementarity and the relative abundance of each oligonucleotidesequence on the micro-array.

[0248] VIII Complementary Polynucleotides

[0249] Sequence complementary to the RCN-encoding sequence, or any partthereof, is used to decrease or inhibit expression of naturallyoccurring RCN. Although use of oligonucleotides comprising from about 15to about 30 base-pairs is described, essentially the same procedure isused with smaller or larger sequence fragments. Appropriateoligonucleotides are designed using OLIGO 4.06 software and the codingsequence of RCN. To inhibit transcription, a complementaryoligonucleotide is designed from the most unique 5′ sequence and used toprevent promoter binding to the coding sequence. To inhibit translation,a complementary oligonucleotide is designed to prevent ribosomal bindingto the RCN-encoding transcript.

[0250] IX Expression of RCN

[0251] Expression of RCN is accomplished by subcloning the cDNAs intoappropriate vectors and transforming the vectors into host cells. Inthis case, the cloning vector is also used to express RCN in E. coli.Upstream of the cloning site, this vector contains a promoter forβ-galactosidase, followed by sequence containing the amino-terminal Met,and the subsequent seven residues of β-galactosidase. Immediatelyfollowing these eight residues is a bacteriophage promoter useful fortranscription and a linker containing a number of unique restrictionsites.

[0252] Induction of an isolated, transformed bacterial strain with IPTGusing standard methods produces a fusion protein which consists of thefirst eight residues of β-galactosidase, about 5 to 15 residues oflinker, and the full length protein. The signal residues direct thesecretion of RCN into the bacterial growth media which can be useddirectly in the following assay for activity.

[0253] X Demonstration of RCN Activity

[0254] The assay for RCN γ and RCN δ is based upon the ability of RCNsto bind Ca²⁺. Ca²⁺ binding is demonstrated directly for RCN using theCa²⁺ overlay system (Weis, K. et al. (supra)). Purified RCN istransferred to nitrocellulose membranes, washed three times with buffer(60 mM KCl, 5 mM MgCl₂, 10 mM imidazole-HCl, pH 6.8), and incubated inthis buffer for 10 minutes with 1 μCi [⁴⁵Ca²⁺] (NEN-DuPont). Unbound[⁴⁵Ca²⁺] is removed by washing with double distilled water, and thedried membranes are autoradiographed using XOMAT film (Kodak).

[0255] XI Production of RCN Specific Antibodies

[0256] RCN that is substantially purified using PAGE electrophoresis(Sambrook, supra), or other purification techniques, is used to immunizerabbits and to produce antibodies using standard protocols. The aminoacid sequence deduced from SEQ ID NO:2 or SEQ ID NO:4 is analyzed usingDNASTAR software (DNASTAR Inc) to determine regions of highimmunogenicity and a corresponding oligopeptide is synthesized and usedto raise antibodies by means known to those of skill in the art.Selection of appropriate epitopes, such as those near the C-terminus orin hydrophilic regions, is described by Ausubel et al. (supra), andothers.

[0257] Typically, the oligopeptides are 15 residues in length aresynthesized using an Applied Biosystems 431 A peptide synthesizer usingfmoc-chemistry, and coupled to keyhole limpet hemocyanin (KLH, Sigma,St. Louis, Mo.) by reaction with N-maleimidobenzoyl-N-hydroxysuccinimideester (MBS; Ausubel et al., supra). Rabbits are immunized with theoligopeptide-KLH complex in complete Freund's adjuvant. The resultingantisera are tested for antipeptide activity, for example, by bindingthe peptide to plastic, blocking with 1% BSA, reacting with rabbitantisera, washing, and reacting with radio iodinated, goat anti-rabbitIgG.

[0258] XII Purification of Naturally Occurring RCN Using SpecificAntibodies

[0259] Naturally occurring or recombinant RCN is substantially purifiedby immunoaffinity chromatography using antibodies specific for RCN. Animmunoaffinity column is constructed by covalently coupling RCN antibodyto an activated chromatographic resin, such as CNBr-activated SEPHAROSE(Pharmacia & Upjohn). After the coupling, the resin is blocked andwashed according to the manufacturer's instructions.

[0260] Media containing RCN is passed over the immunoaffinity column,and the column is washed under conditions that allow the preferentialabsorbance of RCN (e.g., high ionic strength buffers in the presence ofdetergent). The column is eluted under conditions that disruptantibody/RCN binding (eg, a buffer of pH 2-3 or a high concentration ofa chaotrope, such as urea or thiocyanate ion), and RCN is collected.

[0261] XIII Identification of Molecules Which Interact with RCN

[0262] RCN or biologically active fragments thereof are labeled with¹²⁵I Bolton-Hunter reagent (Bolton et al. (1973) Biochem. J. 133: 529).Candidate molecules previously arrayed in the wells of a multi-wellplate are incubated with the labeled RCN, washed and any wells withlabeled RCN complex are assayed. Data obtained using differentconcentrations of RCN are used to calculate values for the number,affinity, and association of RCN with the candidate molecules.

[0263] All publications and patents mentioned in the above specificationare herein incorporated by reference. Various modifications andvariations of the described method and system of the invention will beapparent to those skilled in the art without departing from the scopeand spirit of the invention. Although the invention has been describedin connection with specific preferred embodiments, it should beunderstood that the invention as claimed should not be unduly limited tosuch specific embodiments. Indeed, various modifications of thedescribed modes for carrying out the invention which are obvious tothose skilled in molecular biology or related fields are intended to bewithin the scope of the following claims.

1 6 328 amino acids amino acid single linear RATRNOT02 922578 1 Met MetTrp Arg Pro Ser Val Leu Leu Leu Leu Leu Leu Leu Arg His 1 5 10 15 GlyAla Gln Gly Lys Pro Ser Pro Asp Ala Gly Pro His Gly Gln Gly 20 25 30 ArgVal His Gln Ala Ala Pro Leu Ser Asp Ala Pro His Asp Asp Ala 35 40 45 HisGly Asn Phe Gln Tyr Asp His Glu Ala Phe Leu Gly Arg Glu Val 50 55 60 AlaLys Glu Phe Asp Gln Leu Thr Pro Glu Glu Ser Gln Ala Arg Leu 65 70 75 80Gly Arg Ile Val Asp Arg Met Asp Arg Ala Gly Asp Gly Asp Gly Trp 85 90 95Val Ser Leu Ala Glu Leu Arg Ala Trp Ile Ala His Thr Gln Gln Arg 100 105110 His Ile Arg Asp Ser Val Ser Ala Ala Trp Asp Thr Tyr Asp Thr Asp 115120 125 Arg Asp Gly Arg Val Gly Trp Glu Glu Leu Arg Asn Ala Thr Tyr Gly130 135 140 His Tyr Ala Pro Gly Glu Glu Phe His Asp Val Glu Asp Ala GluThr 145 150 155 160 Tyr Lys Lys Met Leu Ala Arg Asp Glu Arg Arg Phe ArgVal Ala Asp 165 170 175 Gln Asp Gly Asp Ser Met Ala Thr Arg Glu Glu LeuThr Ala Phe Leu 180 185 190 His Pro Glu Glu Phe Pro His Met Arg Asp IleVal Ile Ala Glu Thr 195 200 205 Leu Glu Asp Leu Asp Arg Asn Lys Asp GlyTyr Val Gln Val Glu Glu 210 215 220 Tyr Ile Ala Asp Leu Tyr Ser Ala GluPro Gly Glu Glu Glu Pro Ala 225 230 235 240 Trp Val Gln Thr Glu Arg GlnGln Phe Arg Asp Phe Arg Asp Leu Asn 245 250 255 Lys Asp Gly His Leu AspGly Ser Glu Val Gly His Trp Val Leu Pro 260 265 270 Pro Ala Gln Asp GlnPro Leu Val Glu Ala Asn His Leu Leu His Glu 275 280 285 Ser Asp Thr AspLys Asp Gly Arg Leu Ser Lys Ala Glu Ile Leu Gly 290 295 300 Asn Trp AsnMet Phe Val Gly Ser Gln Ala Thr Asn Tyr Gly Glu Asp 305 310 315 320 LeuThr Arg His His Asp Glu Leu 325 1463 base pairs nucleic acid singlelinear RATRNOT02 922578 2 CGCAGAGCGG ACGTGGAGAG CGGACGNCAG CTGGATAACAGGGGACCGAT GATGTGGCGA 60 CCATCAGTTC TGCTGCTTCT GTTGCTACTG AGGCACGGGGCCCAGGGGAA GCCATCCCCA 120 GACGCAGGCC CTCATGGCCA GGGGAGGGTG CACCAGGCGGCCCCCCTGAG CGACGCTCCC 180 CATGATGACG CCCACGGGAA CTTCCAGTAC GACCATGAGGCTTTCCTGGG ACGGGAAGTG 240 GCCAAGGAAT TCGACCAACT CACCCCAGAG GAAAGCCAGGCCCGTCTGGG GCGGATCGTG 300 GACCGCATGG ACCGCGCGGG GGACGGCGAC GGCTGGGTGTCGCTGGCCGA GCTTCGCGCG 360 TGGATCGCGC ACACGCAGCA GCGGCACATA CGGGACTCGGTGAGCGCGGC CTGGGACACG 420 TACGACACGG ACCGCGACGG GCGTGTGGGT TGGGAGGAGCTGCGCAACGC CACCTATGGC 480 CACTACGCGC CCGGTGAAGA ATTTCATGAC GTGGAGGATGCAGAGACCTA CAAAAAGATG 540 CTGGCTCGGG ACGAGCGGCG TTTCCGGGTG GCCGACCAGGATGGGGACTC GATGGCCACT 600 CGAGAGGAGC TGACAGCCTT CCTGCACCCC GAGGAGTTCCCTCACATGCG GGACATCGTG 660 ATTGCTGAAA CCCTGGAGGA CCTGGACAGA AACAAAGATGGCTATGTCCA GGTGGAGGAG 720 TACATCGCGG ATCTGTACTC AGCCGAGCCT GGGGAGGAGGAGCCGGCGTG GGTGCAGACG 780 GAGAGGCAGC AGTTCCGGGA CTTCCGGGAT CTGAACAAGGATGGGCACCT GGATGGGAGT 840 GAGGTGGGCC ACTGGGTGCT GCCCCCTGCC CAGGACCAGCCCCTGGTGGA AGCCAACCAC 900 CTGCTGCACG AGAGCGACAC GGACAAGGAT GGGCGGCTGAGCAAAGCGGA AATCCTGGGT 960 AATTGGAACA TGTTTGTGGG CAGTCAGGCC ACCAACTATGGCGAGGACCT GACCCGGCAC 1020 CACGATGAGC TGTGAGCACC GCGCACCTGC CACAGCCTCAGAGGCCCGCA CAATGACCGG 1080 AGGAGGGGCC GCTGTGGTCT GGCCCCCTCC CTGTCCAGGCCCCGCAGGAG GCAGATGCAG 1140 TCCCAGGCAT CCTCCTGCCC CTGGGCTCTC AGGGACCCCCTGGGTCGGCT TCTGTCCCTG 1200 TCACACCCCC AACCCCAGGG AGGGGCTGTC ATAGTCCCAGAGGATAAGCA ATACCTATTT 1260 CTGACTGAGT CTCCCAGCCC AGACCCAGGG ACCCTTGGCCCCAAGCTCAG CTCTAAGAAC 1320 CGCCCCAACC CCTCCAGCTC CAAATCTGAG CCTCCACCACATAGACTGAA ACTCCCCTGG 1380 CCCCAGCCCT CTCCTGCCTG GCCTGGCCTG GGACACCTCCTCTCTGCCAG GAGGCAATAA 1440 AAGCCAGCGC CGGGAAAAAA AAA 1463 315 aminoacids amino acid single linear BLADNOT03 1601793 3 Met Asp Leu Arg GlnPhe Leu Met Cys Leu Ser Leu Cys Thr Ala Phe 1 5 10 15 Ala Leu Ser LysPro Thr Glu Lys Lys Asp Arg Val His His Glu Pro 20 25 30 Gln Leu Ser AspLys Val His Asn Asp Ala Gln Ser Phe Asp Tyr Asp 35 40 45 His Asp Ala PheLeu Gly Ala Glu Glu Ala Lys Thr Phe Asp Gln Leu 50 55 60 Thr Pro Glu GluSer Lys Glu Arg Leu Gly Lys Ile Val Ser Lys Ile 65 70 75 80 Asp Gly AspLys Asp Gly Phe Val Thr Val Asp Glu Leu Lys Asp Trp 85 90 95 Ile Lys PheAla Gln Lys Arg Trp Ile Tyr Glu Asp Val Glu Arg Gln 100 105 110 Trp LysGly His Asp Leu Asn Glu Asp Gly Leu Val Ser Trp Glu Glu 115 120 125 TyrLys Asn Ala Thr Tyr Gly Tyr Val Leu Asp Asp Pro Asp Pro Asp 130 135 140Asp Gly Phe Asn Tyr Lys Gln Met Met Val Arg Asp Glu Arg Arg Phe 145 150155 160 Lys Met Ala Asp Lys Asp Gly Asp Leu Ile Ala Thr Lys Glu Glu Phe165 170 175 Thr Ala Phe Leu His Pro Glu Glu Tyr Asp Tyr Met Lys Asp IleVal 180 185 190 Val Gln Glu Thr Met Glu Asp Ile Asp Lys Asn Ala Asp GlyPhe Ile 195 200 205 Asp Leu Glu Glu Tyr Ile Gly Asp Met Tyr Ser His AspGly Asn Thr 210 215 220 Asp Glu Pro Glu Trp Val Lys Thr Glu Arg Glu GlnPhe Val Glu Phe 225 230 235 240 Arg Asp Lys Asn Arg Asp Gly Lys Met AspLys Glu Glu Thr Lys Asp 245 250 255 Trp Ile Leu Pro Ser Asp Tyr Asp HisAla Glu Ala Glu Ala Arg His 260 265 270 Leu Val Tyr Glu Ser Asp Gln AsnLys Asp Gly Lys Leu Thr Lys Glu 275 280 285 Glu Ile Val Asp Lys Tyr AspLeu Phe Val Gly Ser Gln Ala Thr Asp 290 295 300 Phe Gly Glu Ala Leu ValArg His Asp Glu Phe 305 310 315 2658 base pairs nucleic acid singlelinear BLADNOT03 1601793 4 CCCGCTTCCG GTTGGGCGGT GCTTGCGCGC GTGAGCTGAGCCGGTGGGTG AGCGGCGGCC 60 ACGGCATCCT GTGCTGTGGG GGCTACGAGG AAAGATCTAATTATCATGGA CCTGCGACAG 120 TTTCTTATGT GCCTGTCCCT GTGCACAGCC TTTGCCTTGAGCAAACCCAC AGAAAAGAAG 180 GACCGTGTAC ATCATGAGCC TCAGCTCAGT GACAAGGTTCACAATGATGC TCAGAGTTTT 240 GATTATGACC ATGATGCCTT CTTGGGTGCT GAAGAAGCAAAGACCTTTGA TCAGCTGACA 300 CCAGAAGAGA GCAAGGAAAG GCTTGGAAAG ATTGTAAGTAAAATAGATGG CGACAAGGAC 360 GGGTTTGTCA CTGTGGATGA GCTCAAAGAC TGGATTAAATTTGCACAAAA GCGCTGGATT 420 TACGAGGATG TAGAGCGACA GTGGAAGGGG CATGACCTCAATGAGGACGG CCTCGTTTCC 480 TGGGAGGAGT ATAAAAATGC CACCTACGGC TACGTTTTAGATGATCCAGA TCCTGATGAT 540 GGATTTAACT ATAAACAGAT GATGGTTAGA GATGAGCGGAGGTTTAAAAT GGCAGACAAG 600 GATGGAGACC TCATTGCCAC CAAGGAGGAG TTCACAGCTTTCCTGCACCC TGAGGAGTAT 660 GACTACATGA AAGATATAGT AGTACAGGAA ACAATGGAAGATATAGATAA GAATGCTGAT 720 GGTTTCATTG ATCTAGAAGA GTATATTGGT GACATGTACAGCCATGATGG GAATACTGAT 780 GAGCCAGAAT GGGTAAAGAC AGAGCGAGAG CAGTTTGTTGAGTTTCGGGA TAAGAACCGT 840 GATGGGAAGA TGGACAAGGA AGAGACCAAA GACTGGATCCTTCCCTCAGA CTATGATCAT 900 GCAGAGGCAG AAGCCAGGCA CCTGGTCTAT GAATCAGACCAAAACAAGGA TGGCAAGCTT 960 ACCAAGGAGG AGATCGTTGA CAAGTATGAC TTATTTGTTGGCAGCCAGGC CACAGATTTT 1020 GGGGAGGCCT TAGTACGGCA TGATGAGTTC TGAGCTACGGAGGAACCCTC ATTTCCTCAA 1080 AAGTAATTTA TTTTTACAGC TTCTGGTTTC ACATGAAATTGTTTGCGCTA CTGAGACTGT 1140 TACTACAAAC TTTTTAAGAC ATGAAAAGGC GTAATGAAAACCATCCCGTC CCCATTCCTC 1200 CTCCTCTCTG AGGGACTGGA GGGAAGCCGT GCTTCTGAGGAACAACTCTA ATTAGTACAC 1260 TTGTGTTTGT AGATTTACAC TTTGTATTAT GTATTAACATGGCGTGTTTA TTTTTGTATT 1320 TTTCTCTGGT TGGGAGTATG ATATGAAGGA TCAAGATCCTCAACTCACAC ATGTAGACAA 1380 ACATTAGCTC TTTACTCTTT CTCAACCCCT TTTATGATTTTAATAATTCT CACTTAACTA 1440 ATTTTGTAAG CCTGAGATCA ATAAGAAATG TTCAGGAGAGAGGAAAGAAA AAAAATATAT 1500 GCTCCACAAT TTATATTTAG AGAGAGAACA CTTAGTCTTGCCTGTCAAAA AGTCCAACAT 1560 TTCATAGGTA GTAGGGGCCA CATATTACAT TCAGTTGCTATAGGTCCAGC AACTGAACCT 1620 GCCATTACCT GGGCAAGGAA AGATCCCTTT GCTCTAGGAAAGCTTGGCCC AAATTGATTT 1680 TCTTCTTTTT CCCCCTGTAG GACTGACTGT TGGCTAATTTTGTCAAGCAC AGCTGTGGTG 1740 GGAAGAGTTA GGGCCAGTGT CTTGAAAATC AATCAAGTAGTGAATGTGAT CTCTTTGCAG 1800 AGCTATAGAT AGAAACAGCT GGAAAACTAA AGGAAAAATACAAATGTTTT CGGGGCATAC 1860 ATTTTTTTTC TGGGTGTGCA TCTGTTGAAA TGCTCAAGACTTAATTATTT GCCTTTTGAA 1920 ATCACTGTAA ATGCCCCCAT CCGGTTCCTC TTCTTCCCAGGTGTGCCAAG GAATTAATCT 1980 TGGTTTCACT ACAATTAAAA TTCACTCCTT TCCAATCATGTCATTGAAAG TGCCTTTAAC 2040 GAAAGAAATG GTCACTGAAT GGGAATTCTC TTAAGAAACCCTGAGATTAA AAAAAGACTA 2100 TTTGGATAAC TTATAGGAAA GCCTAGAACC TCCCAGTAGAGTGGGGATTT TTTTCTTCTT 2160 CCCTTTCTCT TTTGGACAAT AGTTAAATTA GCAGTATTAGTTATGAGTTT GGTTGCAGTG 2220 TTCTTATCTT GTGGGCTGAT TTCCAAAAAC CACATGCTGCTGAATTTACC AGGGATCCTC 2280 ATACCTCACA ATGCAAACCA CTTACTACCA GGCCTTTTTCTGTGTCCACT GGAGAGCTTG 2340 AGCTCACACT CAAAGATCAG AGGACCTACA GAGAGGGCTCTTTGGTTTGA GGACCATGGC 2400 TTACCTTTCC TGCCTTTGAC CCATCACACC CCATTTCCTCCTCTTTCCCT CTCCCCGCTG 2460 CCAAAAAAAA AAAAAAAGGA AACGTTTATC ATGAATCAACAGGGTTTCAG TCCTTATCAA 2520 AGAGAGATGT GGAAAGAGCT AAAGAAACCA CCCTTTGTTCCCAACTCCAC TTTACCCATA 2580 TTTTATGCAA CACAAACACT GTCCTTTTGG GTCCCTTTCTTACAGATGGG ACCTCTTGAG 2640 GAAGGAATTA TCGTATTC 2658 331 amino acidsamino acid single linear GenBank 1262329 5 Met Ala Arg Gly Gly Arg GlyArg Arg Leu Gly Leu Ala Leu Gly Leu 1 5 10 15 Leu Leu Ala Leu Val LeuAla Pro Arg Val Leu Arg Ala Lys Pro Thr 20 25 30 Val Arg Lys Glu Arg ValVal Arg Pro Asp Ser Glu Leu Gly Glu Arg 35 40 45 Pro Pro Glu Asp Asn GlnSer Phe Gln Tyr Asp His Glu Ala Phe Leu 50 55 60 Gly Lys Glu Asp Ser LysThr Phe Asp Gln Leu Thr Pro Asp Glu Ser 65 70 75 80 Lys Glu Arg Leu GlyLys Ile Val Asp Arg Ile Asp Asn Asp Gly Asp 85 90 95 Gly Phe Val Thr ThrGlu Glu Leu Lys Thr Trp Ile Lys Arg Val Gln 100 105 110 Lys Arg Tyr IlePhe Asp Asn Val Ala Lys Val Trp Lys Asp Tyr Asp 115 120 125 Arg Asp LysAsp Asp Lys Ile Ser Trp Glu Glu Tyr Lys Gln Ala Thr 130 135 140 Tyr GlyTyr Tyr Leu Gly Asn Pro Ala Glu Phe His Asp Ser Ser Asp 145 150 155 160His His Thr Phe Lys Lys Met Leu Pro Arg Asp Glu Arg Arg Phe Lys 165 170175 Ala Ala Asp Leu Asn Gly Asp Leu Thr Ala Thr Arg Glu Glu Phe Thr 180185 190 Ala Phe Leu His Pro Glu Glu Phe Glu His Met Lys Glu Ile Val Val195 200 205 Leu Glu Thr Leu Glu Asp Ile Asp Lys Asn Gly Asp Gly Phe ValAsp 210 215 220 Gln Asp Glu Tyr Ile Ala Asp Met Phe Ser His Glu Glu AsnGly Pro 225 230 235 240 Glu Pro Asp Trp Val Leu Ser Glu Arg Glu Gln PheAsn Glu Phe Arg 245 250 255 Asp Leu Asn Lys Asp Gly Lys Leu Asp Lys AspGlu Ile Arg His Trp 260 265 270 Ile Leu Pro Gln Asp Tyr Asp His Ala GlnAla Glu Ala Arg His Leu 275 280 285 Val Tyr Glu Ser Asp Lys Asn Lys AspGlu Lys Leu Thr Lys Glu Glu 290 295 300 Ile Leu Glu Asn Trp Asn Met PheVal Gly Ser Gln Ala Thr Asn Tyr 305 310 315 320 Gly Glu Asp Leu Thr LysAsn His Asp Glu Leu 325 330 98 amino acids amino acid single linearGenBank 780361 6 Arg Arg Asp Val Ala Lys Glu Phe Asp Gln Leu Thr Pro GluGlu Ser 1 5 10 15 Gln Ala Arg Leu Gly Arg Ile Val Asp Arg Met Asp LeuAla Gly Asp 20 25 30 Ser Asp Gly Trp Val Ser Leu Ala Ala Leu Arg Ala TrpIle Ala His 35 40 45 Thr Gln Gln Arg His Ile Arg Asp Ser Val Ser Ala AlaTrp His Thr 50 55 60 Tyr Asp Thr Asp Arg Asp Gly Arg Val Gly Trp Glu GluLeu Arg Asn 65 70 75 80 Ala Thr Tyr Gly His Tyr Glu Pro Gly Glu Glu PheHis Asp Val Glu 85 90 95 Gly Pro

What is claimed is:
 1. An isolated polypeptide selected from the groupconsisting of: a) a polypeptide comprising an amino acid sequence of SEQID NO:1 or SEQ ID NO:3, b) a naturally occurring polypeptide comprisingan amino acid sequence at least 90% identical to the amino acid sequenceof SEQ ID NO:1 or SEQ ID NO:3, c) a biologically active fragment of apolypeptide having the amino acid sequence of SEQ ID NO:1 or SEQ IDNO:3, and d) an immunogenic fragment of a polypeptide having the aminoacid sequence of SEQ ID NO:1 or SEQ ID NO:3.
 2. An isolated polypeptideof claim 1 selected from the group consisting of SEQ ID NO:1 and SEQ IDNO:3.
 3. An isolated polynucleotide encoding a polypeptide of claim 1.4. An isolated polynucleotide encoding a polypeptide of claim
 2. 5. Anisolated polynucleotide of claim 4 selected from the group consisting ofSEQ ID NO:2 and SEQ ID NO:4.
 6. A recombinant polynucleotide comprisinga promoter sequence operably linked to a polynucleotide of claim
 3. 7. Acell transformed with a recombinant polynucleotide of claim
 6. 8. Atransgenic organism comprising a recombinant polynucleotide of claim 6.9. A method for producing a polypeptide of claim 1, the methodcomprising: a) culturing a cell under conditions suitable for expressionof the polypeptide, wherein said cell is transformed with a recombinantpolynucleotide, and said recombinant polynucleotide comprises a promotersequence operably linked to a polynucleotide encoding the polypeptide ofclaim 1, and b) recovering the polypeptide so expressed.
 10. An isolatedantibody which specifically binds to a polypeptide of claim
 1. 11. Anisolated polynucleotide selected from the group consisting of: a) apolynucleotide comprising a polynucleotide sequence of SEQ ID NO:2 orSEQ ID NO:4, b) a naturally occurring polynucleotide comprising apolynucleotide sequence at least 90% identical to the polynucleotidesequence of SEQ ID NO:2 or SEQ ID NO:4, c) a polynucleotidecomplementary to a polynucleotide of a), d) a polynucleotidecomplementary to a polynucleotide of b), and e) an RNA equivalent ofa)-d).
 12. An isolated polynucleotide comprising at least 60 contiguousnucleotides of a polynucleotide of claim
 11. 13. A method for detectinga target polynucleotide in a sample, said target polynucleotide having asequence of a polynucleotide of claim 11, the method comprising: a)hybridizing the sample with a probe comprising at least 20 contiguousnucleotides comprising a sequence complementary to said targetpolynucleotide in the sample, and which probe specifically hybridizes tosaid target polynucleotide, under conditions whereby a hybridizationcomplex is formed between said probe and said target polynucleotide orfragments thereof, and b) detecting the presence or absence of saidhybridization complex, and, optionally, if present, the amount thereof.14. A method of claim 13, wherein the probe comprises at least 60contiguous nucleotides.
 15. A method for detecting a targetpolynucleotide in a sample, said target polynucleotide having a sequenceof a polynucleotide of claim 11, the method comprising: a) amplifyingsaid target polynucleotide or fragment thereof using polymerase chainreaction amplification, and b) detecting the presence or absence of saidamplified target polynucleotide or fragment thereof, and, optionally, ifpresent, the amount thereof.
 16. A composition comprising a polypeptideof claim 1 and a pharmaceutically acceptable excipient.
 17. Acomposition of claim 16, wherein the polypeptide has an amino acidsequence of SEQ ID NO:1 or SEQ ID NO:3.
 18. A method for treating adisease or condition associated with decreased expression of functionalHMRP, comprising administering to a patient in need of such treatmentthe composition of claim
 16. 19. A method for screening a compound foreffectiveness as an agonist of a polypeptide of claim 1, the methodcomprising: a) exposing a sample comprising a polypeptide of claim 1 toa compound, and b) detecting agonist activity in the sample.
 20. Acomposition comprising an agonist compound identified by a method ofclaim 19 and a pharmaceutically acceptable excipient.
 21. A method fortreating a disease or condition associated with decreased expression offunctional HMRP, comprising administering to a patient in need of suchtreatment a composition of claim
 20. 22. A method for screening acompound for effectiveness as an antagonist of a polypeptide of claim 1,the method comprising: a) exposing a sample comprising a polypeptide ofclaim 1 to a compound, and b) detecting antagonist activity in thesample.
 23. A composition comprising an antagonist compound identifiedby a method of claim 22 and a pharmaceutically acceptable excipient. 24.A method for treating a disease or condition associated withoverexpression of functional HMRP, comprising administering to a patientin need of such treatment a composition of claim
 23. 25. A method ofscreening for a compound that specifically binds to the polypeptide ofclaim 1, said method comprising the steps of: a) combining thepolypeptide of claim 1 with at least one test compound under suitableconditions, and b) detecting binding of the polypeptide of claim 1 tothe test compound, thereby identifying a compound that specificallybinds to the polypeptide of claim
 1. 26. A method of screening for acompound that modulates the activity of the polypeptide of claim 1, saidmethod comprising: a) combining the polypeptide of claim 1 with at leastone test compound under conditions permissive for the activity of thepolypeptide of claim 1, b) assessing the activity of the polypeptide ofclaim 1 in the presence of the test compound, and c) comparing theactivity of the polypeptide of claim 1 in the presence of the testcompound with the activity of the polypeptide of claim 1 in the absenceof the test compound, wherein a change in the activity of thepolypeptide of claim 1 in the presence of the test compound isindicative of a compound that modulates the activity of the polypeptideof claim
 1. 27. A method for screening a compound for effectiveness inaltering expression of a target polynucleotide, wherein said targetpolynucleotide comprises a sequence of claim 5, the method comprising:a) exposing a sample comprising the target polynucleotide to a compound,under conditions suitable for the expression of the targetpolynucleotide, b) detecting altered expression of the targetpolynucleotide, and c) comparing the expression of the targetpolynucleotide in the presence of varying amounts of the compound and inthe absence of the compound.
 28. A method for assessing toxicity of atest compound, said method comprising: a) treating a biological samplecontaining nucleic acids with the test compound; b) hybridizing thenucleic acids of the treated biological sample with a probe comprisingat least 20 contiguous nucleotides of a polynucleotide of claim 11 underconditions whereby a specific hybridization complex is formed betweensaid probe and a target polynucleotide in the biological sample, saidtarget polynucleotide comprising a polynucleotide sequence of apolynucleotide of claim 11 or fragment thereof; c) quantifying theamount of hybridization complex; and d) comparing the amount ofhybridization complex in the treated biological sample with the amountof hybridization complex in an untreated biological sample, wherein adifference in the amount of hybridization complex in the treatedbiological sample is indicative of toxicity of the test compound.
 29. Adiagnostic test for a condition or disease associated with theexpression of HMRP in a biological sample comprising the steps of: a)combining the biological sample with an antibody of claim 10, underconditions suitable for the antibody to bind the polypeptide and form anantibody:polypeptide complex; and b) detecting the complex, wherein thepresence of the complex correlates with the presence of the polypeptidein the biological sample.
 30. The antibody of claim 10, wherein theantibody is: a) a chimeric antibody, b) a single chain antibody, c) aFab fragment, d) a F(ab′)₂ fragment, or e) a humanized antibody.
 31. Acomposition comprising an antibody of claim 10 and an acceptableexcipient.
 32. A method of diagnosing a condition or disease associatedwith the expression of HMRP in a subject, comprising administering tosaid subject an effective amount of the composition of claim
 31. 33. Acomposition of claim 31, wherein the antibody is labeled.
 34. A methodof diagnosing a condition or disease associated with the expression ofHMRP in a subject, comprising administering to said subject an effectiveamount of the composition of claim
 33. 35. A method of preparing apolyclonal antibody with the specificity of the antibody of claim 10comprising: a) immunizing an animal with a polypeptide having an aminoacid sequence of SEQ ID NO:1 or SEQ ID NO:3, or an immunogenic fragmentthereof, under conditions to elicit an antibody response; b) isolatingantibodies from said animal; and c) screening the isolated antibodieswith the polypeptide, thereby identifying a polyclonal antibody whichbinds specifically to a polypeptide having an amino acid sequence of SEQID NO:1 or SEQ ID NO:3.
 36. An antibody produced by a method of claim35.
 37. A composition comprising the antibody of claim 36 and a suitablecarrier.
 38. A method of making a monoclonal antibody with thespecificity of the antibody of claim 10 comprising: a) immunizing ananimal with a polypeptide having an amino acid sequence of SEQ ID NO:1or SEQ ID NO:3, or an immunogenic fragment thereof, under conditions toelicit an antibody response; b) isolating antibody producing cells fromthe animal; c) fusing the antibody producing cells with immortalizedcells to form monoclonal antibody-producing hybridoma cells; d)culturing the hybridoma cells; and e) isolating from the culturemonoclonal antibody which binds specifically to a polypeptide having anamino acid sequence of SEQ ID NO:1 or SEQ ID NO:3.
 39. A monoclonalantibody produced by a method of claim
 38. 40. A composition comprisingthe antibody of claim 39 and a suitable carrier.
 41. The antibody ofclaim 10, wherein the antibody is produced by screening a Fab expressionlibrary.
 42. The antibody of claim 10, wherein the antibody is producedby screening a recombinant immunoglobulin library.
 43. A method fordetecting a polypeptide having an amino acid sequence of SEQ ID NO:1 orSEQ ID NO:3 in a sample, comprising the steps of: a) incubating theantibody of claim 10 with a sample under conditions to allow specificbinding of the antibody and the polypeptide; and b) detecting specificbinding, wherein specific binding indicates the presence of apolypeptide having an amino acid sequence of SEQ ID NO:1 or SEQ ID NO:3in the sample.
 44. A method of purifying a polypeptide having an aminoacid sequence of SEQ ID NO:1 or SEQ ID NO:3 from a sample, the methodcomprising: a) incubating the antibody of claim 10 with a sample underconditions to allow specific binding of the antibody and thepolypeptide; and b) separating the antibody from the sample andobtaining the purified polypeptide having an amino acid sequence of SEQID NO:1 or SEQ ID NO:3.
 45. A polypeptide of claim 1, comprising theamino acid sequence of SEQ ID NO:1.
 46. A polypeptide of claim 1,comprising the amino acid sequence of SEQ ID NO:3.
 47. A polynucleotideof claim 11, comprising the polynucleotide sequence of SEQ ID NO:2. 48.A polynucleotide of claim 11, comprising the polynucleotide sequence ofSEQ ID NO:4.